JPH06102013B2 - Bioreactor - Google Patents

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 aeration gas supply, contact between cells and bubbles due to bubbling due to aeration bubbling into the medium and mechanical stress due to agitation to the cells There is a problem that cells, particularly fragile cells such as animal cells, may be killed and damaged due to addition.

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 provided separately. As shown in FIG. 3, the pH and DO of the medium before (or after) the bioreactor 14 are
Measured by the pH sensor 13 and the DO sensor 18, gas is sent from the gas exchanger 17 according to the signal, but when the cells grow rapidly, the medium to be put into the bioreactor 14
The pH value and DO value 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 in which O ₂ gas is sufficiently supplied not only to the medium but also to the cell. It is intended to provide a bioreactor suitable for culturing. And, according to the present invention, the purpose thereof is to provide 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. And a bioreactor characterized by accommodating a large number of porous hollow fiber membranes forming the medium supply membrane and a large number of porous hollow fiber membranes forming a gas exchange membrane in a mixed state. be able to.

[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 pores of the membrane,
Grow the cells that are seeded on the outside of the media feeding membrane.
At that time, similarly, the gas sent through the inside of the gas exchange membrane housed in the bioreactor mixed with the culture medium supply membrane is the medium oozing out from the culture medium supply membrane through the gas exchange membrane. In addition to supplying O ₂ to the medium, it also supplies O ₂ to cells planted outside the medium supply membrane.

[Examples] Hereinafter, the present invention will be described in detail based on examples of 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 support member 10 made of a potting material such as polyurethane resin, and the medium supply film 8
And both ends of the gas exchange membrane 9 are open to both ends of the cylindrical 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 outside thereof, and each has a large number of through holes 12 through which gas and liquid can pass. The partition walls 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 to keep 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 gas exchange membrane 9 and the through holes 12 of the partition wall 11, 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 polymer may be any of various polymers, and examples of the polymer include cellulose acetate, polysulfone, polyacrylonitrile, fluoropolymer, and polyolefin.

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

Further, it is desirable that the culture medium supply membrane 8 has the ability to permeate water to some extent. That is, it is advantageous that the medium supply membrane 8 has a water permeability coefficient (ml / m ² · hr · mmHg) of 10 or more, preferably 100 or more. On the other hand, there is no particular upper limit, but 20,0
00 or less, preferably 10,000 or less is desired.

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. In FIG. 1, for the sake of convenience of description, the gas exchange membrane 9 is provided in the central portion and the culture medium supply membrane 8 is provided outside the same, and each is separated by the partition wall 11. In order to sufficiently supply the medium, it is more preferable that the tubular container 7 has the gas exchange membrane 9 and the medium supply membrane 8 mixed therein.

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, which comprises a bioreactor 14 equipped with a pH sensor 13 and a DO (dissolved oxygen) sensor 18, a medium tank 15,
1 shows a system consisting of a pH sensor and a pump 16 that controls the flow rate of perfusate. On the other hand, FIG. 3 shows a cell culture system using a conventional bioreactor and a gas exchanger. The difference from FIG. 2 is that the gas exchanger 17 is not included in the bioreactor 14, The point is that it 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 membrane, an ultrafiltration hollow fiber membrane 2,500 made of polysulfone having an inner diameter of 300 μm, an outer diameter of 400 μm, and a molecular weight cutoff of 10,000.
As the gas exchange membrane, a bioreactor consisting of 2,500 polypropylene porous hollow fiber membranes having an inner diameter of 300 μm, an outer diameter of 400 μm, an average pore diameter of 0.2 μm and a porosity of 65% was used.
(Effective membrane area is 0.5m ² for both.) Bioreactor 14 and pH sensor 13, DO (dissolved oxygen) sensor 18, CO ₂ sensor (not shown), medium tank 15, pump 16 are made of silicone tube. connection,
It was a closed circuit. The inside of the circuit was primed, and the whole circuit was subjected to high-pressure steam sterilization for 25 minutes.

After sterilization, the circuit was placed in a constant temperature bath at 37 ° C, and serum-free Eagle's MEM-E (Minimu) was used as a basal medium.
m Essential Medium-Eagle) medium (penicillin potassium
(Including 100,000 units / L, kanamycin sulfate 100 mg / L) 80 ml /
After circulating for 48 hours at min, 10% FBS (Fetal Bovine Ser
um) (serum of calf).

HeLa cells were inoculated (inoculated) with 5 × 10 ⁶ cells / ml in the space outside the hollow fiber membrane. After inoculation, the bioreactor was rotated by 90 degrees every hour without circulating the medium for 4 hours, and HeLa cells were uniformly dispersed in the space outside the hollow fiber membrane. Then, the medium was circulated at 40 ml / min for 4 hours, and then the flow rate was increased to 80 ml / min. Similarly, air (1000 cc / min) and carbon dioxide (50 cc / min) were flown into the gas exchange membrane so that the pH of the medium for cell growth was maintained at 7.4.

The 2 l medium 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. From the 16th day, the medium was replaced every 2 days, and the apparatus was stopped after 31 days.

After stopping the device, PBS (phosphate buffer solution) inside the culture medium supply membrane
(-), Replace with 0.25% trypsin sequentially, 15 minutes each 37 ℃
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 (−) and then with 1%. It was dyed with osninic acid, ligated and dried, and after vapor deposition of gold, SEM (scanning electron microscope) observation was performed.

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 40 mg / dl in 3 days. After that, 1
The measurement every two days from the 6th day showed an almost constant decrease from 100 mg / dl to 50 mg / dl.

Oxygen partial pressure and carbon dioxide partial pressure were almost constant at about 150 and 20 mmHg, respectively, and pH fluctuations were stable between 7.36 and 7.46.

The number of cells cultured for 31 days was 3 × 10 ⁸ cells / ml.

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 used, using the same medium supply membrane and gas exchange membrane used in the examples.

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.

In addition, the pH changes drastically between 5.83 and 8.14, and the oxygen partial pressure also changes drastically.

The fact that the glucose concentration did not change from the 15th day was due to cell death, and when the oxygen partial pressure in the bioreactor was measured, it 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.

As described [Effect INVENTION above in detail, according to the bioreactor of the present invention, since the housing in a state of being mixed with the gas exchange membrane with medium supply film in the bioreactor, O ₂ gas and
The pH adjusting gas is sufficiently supplied to the medium and the cells, and the cells can be grown and proliferated without any trouble, and it has an advantage that it can be sufficiently applied to a large-scale cell culture.

[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 ...
Support member, 11 ... Partition wall, 12 ... Through hole, 13 ... pH sensor, 14 ...
Bioreactor, 17 ... gas exchanger, 18 ... DO sensor.

Claims (3)

[Claims] 1. In 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 the space outside the medium supply membrane, the medium supply membrane is provided in the bioreactor. A bioreactor comprising a large number of porous hollow fiber membranes and a large number of porous hollow fiber membranes forming a gas exchange membrane in a mixed state. 2. The bioreactor according to claim 1, wherein the gas exchange membrane is a polyolefin porous hollow fiber membrane. 3. The bioreactor according to claim 1, which is made of a material that can be sterilized by high-temperature high-pressure steam.