JPH0659206B2 - Bioreactor - Google Patents

Abstract

PURPOSE:To meet the cell culture of a large scale by providing the bioreactor with the membrane for feeding the culture medium and the other membrane for exchanging the gases using porous hollow fiber membranes which are made hydrophilic by treatment as the membrane for feeding culture medium to make it possible to feed a sufficient amount of oxygen gas to the culture medium and the cells. CONSTITUTION:The culture medium is fed through the membrane 8 and the cells are proliferated on the membrane 8 and the outer space of the membrane 8. In addition, the bioreactor 14 is provided with the other membrane 9 for exchanging gases inside, while the membrane 8 for supplying the medium is constituted with porous hollow fibers which is made hydrophilic by treatment. As a result, sufficient amount of oxygen-containing gas and pH-controlling gas are supplied sufficiently to the culture medium and the cells to grow and proliferate the cells without any trouble whereby the process according the present invention can meet satisfactorily a large scale of cell culture.

Description

TECHNICAL FIELD The present invention relates to a bioreactor for growing cells, and more particularly to a bioreactor for culturing cells on a large scale.

[Prior Art] In a culture device using a culture tank for culturing microorganisms or cells, it is known to increase the supply of aeration gas to the medium as one of the means for increasing the volumetric efficiency of the culture. In this case, vigorous stirring of the medium is generally performed.

On the other hand, there is known a culture method called a hollow fiber method, in which cells are cultured using a hollow fiber membrane.

[Problems to be Solved by the Invention] However, in the method of increasing the supply of aeration gas, cells are brought into contact with each other due to foaming caused by aeration bubbling into the medium, and mechanical stress due to stirring is applied to the cells. Therefore, there is a problem that cells, particularly fragile cells such as animal cells, may be killed and damaged.

On the other hand, in the culture method called the hollow fiber method, since the hollow fiber bioreactor and the gas exchanger are separately provided as shown in FIG. 3, the supply of O ₂ and pH adjusting gas is not performed. There is a problem that it is sufficient and not suitable for large-scale cell culture. Explaining this point, in the hollow fiber bioreactor, it is necessary to send a strictly controlled medium to the cells outside the hollow fiber membrane, but the bioreactor 14 and the gas exchanger 17 are required.
When the two are separately provided, as shown in FIG.
Before (or after) the bioreactor 14, p
H and DO are measured by the pH sensor 13 and the DO sensor 18, and gas is sent from the gas exchanger 17 according to the signal, but when the cells grow rapidly, the pH value of the medium that enters the bioreactor 14, DO The values will be different. This is because the cells consume glucose and the like in the medium. Therefore, even if the pH value and the DO value are measured in front of the bioreactor 14, the values are merely approximate values, and it is meaningless to perform gas exchange based on the signals of the values.

Furthermore, in the case of the conventional hollow fiber method, since O ₂ is supplied only to the medium, there is a problem that cells become deficient in oxygen and die.

[Means for Solving the Problems] Therefore, the present invention has been made in view of the above-mentioned drawbacks of the prior art, and a large-scale cell culture in which O ₂ gas is sufficiently supplied not only to the medium but also to the cells. The purpose of the present invention is to provide a bioreactor suitable for. And, according to the present invention, the purpose thereof is to supply a culture solution through a medium supply membrane, and in a bioreactor for growing cells on the medium supply membrane and in an outer space of the medium supply membrane, in the bioreactor. This can be achieved by a bioreactor characterized in that a gas exchange membrane is provided together with the culture medium supply membrane, and the culture medium supply membrane is composed of a hydrophilic hollow fiber membrane.

[Operation] In the bioreactor of the present invention, the medium is flown inside the medium supply membrane arranged in the bioreactor, and the medium is exuded to the outside of the medium supply membrane through the cells of the membrane,
Grow the cells that are seeded on the outside of the media feeding membrane.
At that time, in the same manner, the gas sent through the inside of the gas exchange membrane disposed together with the medium supply membrane in the bioreactor is brought into contact with the medium that has exuded from the medium supply membrane through the gas exchange membrane. turned in and supplies addition, the O ₂ in cells implanted on the outside of the medium supply film O ₂ is supplied to the medium.

Further, since the culture medium supply membrane of the present invention is hydrophilized, the amount of water permeation can be increased.

[Examples] Hereinafter, the present invention will be described in detail based on illustrated examples.

FIG. 1 is a vertical cross-sectional view showing an example of the structure of the bioreactor of the present invention. Inside the cylindrical container 7, a medium supply film 8 is provided.
And a large number of hollow fiber membranes constituting the gas exchange membrane 9 are provided, and the inner surface of the tubular container 7 and the outer surfaces of the medium supply membrane 8 and the gas exchange membrane 9 are provided at both ends of the tubular container 7. Is airtightly supported by a supporting member 10 made of a potting material such as polyurethane resin, and both ends of the medium supply membrane 8 and the gas exchange membrane 9 are open to both ends of the tubular container 7.

The medium supply membrane 8 and the gas exchange membrane 9 are arranged in the cylindrical container 7 such that the gas exchange membrane 9 is arranged at the center and the medium supply membrane 8 is arranged on the outer side thereof, and a large number of through holes 12 through which gas and liquid can pass. The partitions 11 are provided so as to be separated from each other.

Port 5 is a port for taking out the used nutrient medium and cell products from the bioreactor.
Is an inlet of a sensor that controls the supply amount and concentration of gas. The O-ring 19 is provided in order to maintain the airtightness with the outside and to prevent the gas and the medium from being mixed.

In the above configuration, the medium A enters the hollow fiber membrane of the medium supply membrane 8 from the medium inlet 2, and oozes out to the outside of the small amount medium supply membrane 8 due to the pressure applied to the membrane, and the remaining medium from the medium outlet 3 into a cylinder. Exit the container 7. On the other hand, gas B such as air
Enters the hollow fiber membrane of the gas exchange membrane 9 from the gas inlet 1.
Gas is supplied to the culture medium through the through holes 12 of the partition wall 11 through the gas exchange membrane 9, and the residual gas and the exchanged gas exit the tubular container 7 from the gas outlet 4.

Medium supply membrane 8 constituting the bioreactor of the present invention
The substrate is a porous hollow fiber made of various polymers, and examples of the polymer include cellulose acetate, polysulfone, polyacrylonitrile, fluoropolymers, polyolefins (polypropylene, polyethylene, etc.) and the like.

The wall membrane of the porous hollow fiber membrane, which is the medium supply membrane 8, is provided with a large number of cells having a size that allows nutrients, cell waste products, metabolic products, etc. to pass through but does not pass through. When the cells themselves are suspended and cultured, the size of the pores is determined by the size of the cells, but generally, the average pore diameter is 10 μm or less, preferably 8 μm or less.

Further, since the medium supply film 8 is hydrophilized,
The amount of water permeation can be increased.

Examples of the hydrophilic treatment method include the following.

Carboxymethyl ethyl cellulose, glycerin fatty acid ester, hydroxypropyl methyl cellulose acetate succinate, hydroxypropyl methyl cellulose phthalate, poly (2-hydroxyethyl methacrylate), ethylene oxide graft nylon , 6/66
/ 610 Nylon terpolymer, 8-type soluble nylon (methoxy nylon) and the like.

That is, the medium supply membrane 8 has a water permeability coefficient (ml / m ² · hr · mmH
It is preferable that g) is 10 or more, preferably 100 or more. On the other hand, there is no particular upper limit, but 20,000 or less,
It is preferably 10,000 or less.

Further, as the medium supply membrane 8, it is also possible to use a membrane which allows compounds having a small molecular weight such as nutrients and waste products of cells to permeate but does not allow compounds having a large molecular weight to permeate, for example, an ultrafiltration membrane.

Gas exchange membrane 9 constituting the bioreactor of the present invention
It may be made of various polymers, and examples thereof include cellulose, polyacrylonitrile, polycarbonate, polyvinylidene fluoride, polymethylmethacrylate, polyethylene, polypropylene, silicone rubber and modified materials thereof.

Further, the gas exchange membrane 9 needs to have gas permeability. That is, the gas exchange membrane 9 has a gas permeability coefficient (ml / m ²
-Hr · mmHg) is advantageously 10 or more, preferably 100 or more.

Further, the gas exchange membrane 9 is preferably a hollow fiber membrane that can reduce the membrane area per unit volume. In particular, a porous membrane having high permeability is preferable, and among them, a polyolefin-based porous membrane is preferable.

Note that, in FIG. 1, for convenience of description, they are concentric,
A gas exchange membrane 9 is provided in the center of the medium, and a culture medium supply membrane 8 is provided outside the gas exchange membrane, and the culture medium supply membrane 8 is separated by a partition wall 11. It is more preferable that the exchange membrane 9 and the medium supply membrane 8 are mixed.

Further, in order to grow cells, it is absolutely necessary to eliminate bacterial contamination, and the gas exchange membrane 9 is a polypropylene porous membrane, the medium supply membrane 8 is a polyethersulfone ultrafiltration membrane, a tubular shape. It is more preferable to use a polycarbonate container as the container 7 and a polyurethane resin or the like as the support member 10 so that the entire bioreactor can be sterilized by high-temperature high-pressure steam.

FIG. 2 shows a cell culture system using the bioreactor of the present invention. The bioreactor 14 equipped with a pH sensor 13 and a DO (dissolved oxygen) sensor 18, a culture medium tank 15, a pH sensor and a perfusate flow rate. 1 shows a system consisting of a pump 16 for controlling the. On the other hand, FIG. 3 shows a cell culture system using a conventional bioreactor and a gas exchanger. What is different from FIG. 2 is the gas exchanger 1
7 is not contained in the bioreactor 14 but is installed outside.

Therefore, the results of carrying out the present invention with reference to FIGS. 2 and 3 will be described below in more detail.

(Example) As a medium supply film, an inner diameter of 300 μm, an outer diameter of 400 μm,
2,500 polypropylene porous hollow fiber membranes coated with glycerin resin acid ester having an average pore diameter of 0.3 μm and a porosity of 68%, an inner diameter of 300 μ as a gas exchange membrane
m, outer diameter 400 μm, average pore diameter 0.2 μm, porosity 65
A bioreactor consisting of 2,500% polypropylene porous hollow fiber membranes was used. (Effective membrane area is 0.5 m ² for both.) Bioreactor 14 and pH sensor 13, DO (dissolved oxygen) sensor 18, CO ₂ sensor (not shown) and medium tank 15, pump 16 in silicone tube. Connected to form a closed circuit. Prime the circuit,
All circuits were subjected to high-pressure steam sterilization for 25 minutes without any change.

After sterilization, the circuit was placed in a constant temperature bath at 37 ° C, and serum-free Eagle's MEM-E (Minimum
Essential Medium-Eagle) medium (including 100,000 units of penicillin potassium / L and 100 mg / L of kanamycin sulfate)
Was circulated at 80 ml / min for 48 hours, then 10% FBS (Fet
al Bovine Serum) (calf serum) was replaced with MEM-E medium.

5 × 10 ⁶ HeLa cells in the space outside the hollow fiber membrane
Cells / ml were inoculated. After inoculation, the medium was not circulated for 4 hours, and the bioreactor was rotated by 90 degrees every hour to uniformly disperse HeLa cells in the space outside the hollow fiber membrane. afterwards,
After circulating the medium at 40 ml / min for 4 hours, the flow rate was increased to 80 ml / min. Similarly, air (1000 cc / min) and carbon dioxide (50 cc / mi) were added to the cell growth medium to maintain the pH at 7.4.
n) flowed inside the gas exchange membrane.

The medium of No. 2 was replaced every 3 days for 15 days, and the glucose concentration, oxygen partial pressure, and carbon dioxide partial pressure in the medium tank were measured. 1
From the 6th day, the medium was replaced every 2 days, and the apparatus was stopped after 31 days.

After stopping the device, PBS (phosphate buffer) (-),
Subsequent replacement with 0.25% trypsin, 37 ° C for 15 minutes each
After culturing in, the cells were collected by floating and the number of cells was calculated. Similarly, the inside of the medium supply membrane was sequentially replaced with PBS (-) and 2% glutaraldehyde, fixed with 2.5% glutaraldehyde for one day and then washed with PBS (-), 1%
It was dyed with osninic acid, freeze-dried thereafter, and subjected to SEM (scanning electron microscope) observation after gold deposition.

The glucose concentration of the medium was adjusted to an initial value of 100 mg / dl. No remarkable decrease in glucose concentration was observed for 6 days (two medium changes) after the start of culture.

A gradual decrease in glucose concentration was observed from the 9th day, and on the 15th day, it decreased from 100 mg / dl to 20 mg / dl in 3 days.
After that, 100 mg / dl was measured every two days from the 16th day.
To 30 mg / dl, the decrease was almost constant.

Oxygen partial pressure and carbon dioxide partial pressure are about 150 and 20 mmHg from beginning to end.
And the pH value varies from 7.36 to 7.40.
Was stable between.

Number of cells in 31-day culture is 6 × 10 ⁸ cells / ml
Met.

According to SEM observation, the cells attached to the hollow fiber membrane entered the inside of the membrane and grew, and also grew outward. The cells that entered the inside of the membrane did not show much three-dimensional growth, but in the cells that grew toward the outside of the membrane, the cells were adjacent to each other and formed into a three-dimensional structure formed in vivo. It showed similar growth.

(Comparative Example) A bioreactor and a gas exchanger each having an effective membrane area of 0.5 m ² were separately prepared as shown in FIG. 3 by using the same medium supply membrane and gas exchange membrane used in the examples. used.

Sterilization, priming, and cell culture were performed in the same manner as in the examples.

As a result, the glucose concentration gradually decreased from day 9 as in the example, but from day 15 the glucose concentration did not change from the initial value of 100 mg / dl.

Further, the fluctuation of pH fluctuated drastically between 5.83 and 8.14, and the oxygen partial pressure fluctuated similarly.

The glucose concentration did not change from the 15th day because the cells died, and the oxygen partial pressure in the bioreactor was almost 0.

It is presumed that the dissolved oxygen in the medium in the bioreactor was consumed along with the cell growth, the oxygen was almost lost, and the cells were killed.

[Effects of the Invention] As described in detail above, according to the bioreactor of the present invention, a gas exchange membrane is arranged together with a medium supply membrane in the bioreactor, and the hydrophilicity-improved porosity is used as the medium supply membrane. Since a hollow fiber membrane is used, O ₂ gas and gas for pH adjustment are sufficiently supplied to the medium and cells, cell growth and proliferation can be performed without any trouble, and it is also possible to cope with large-scale cell culture. , Has the advantage.

[Brief description of drawings]

FIG. 1 is a longitudinal sectional view showing an example of the structure of the bioreactor of the present invention, FIG. 2 is a schematic view showing a cell culture system using the bioreactor of the present invention, and FIG. 3 is a conventional bioreactor and gas. It is a schematic diagram showing a cell culture system using an exchanger. 7 ... Cylindrical container, 8 ... Medium supply membrane, 9 ... Gas exchange membrane, 10 ... Supporting member, 11 ... Partition wall, 12 ... Through hole,
13 ... pH sensor, 14 ... Bioreactor, 17
...... Gas exchanger, 18 ... DO sensor.

Claims (1)

[Claims] 1. A bioreactor in which a culture solution is supplied through a medium supply membrane and cells are grown on the medium supply membrane and in an outer space of the medium supply membrane, in the bioreactor together with the medium supply membrane, a gas. A bioreactor in which a replacement membrane is provided, and the medium supply membrane is a hydrophilic hollow fiber membrane.