JPH0657141B2 - Hollow fiber membrane cell culture device - Google Patents

Description

TECHNICAL FIELD The present invention relates to a hollow fiber membrane cell culture device that can be heat-sterilized in a high temperature and high pressure steam atmosphere.

The hollow fiber membrane cell culture device referred to here is for controlling the flow rate of the hollow fiber membrane cell culture unit, culture medium reservoir, gas exchanger, pH meter, DO (dissolved oxygen) meter, and perfusate. The pump is set with a tube to make a closed circuit.

[Prior Art] When culturing cells in a cell culture device, it is necessary to provide the device in a sterile state before culturing the cells. Conventionally, as a sterilization method, a pH meter, a DO
All units except the meter are sterilized with ethylene oxide, then exposed to air and exposed to the perfusing medium.
A method of treating the tip of a meter or a DO meter with 70% ethanol is used.

[Problems to be Solved by the Invention] However, in the conventional ethylene oxide gas sterilization method, the cell culturing device is arranged in an atmosphere of 10 to 27% ethylene oxide gas. Toxicity becomes a problem and must be exposed to air for a long time. Also, with ethanol, pH meter, DO
Even if the tip of the meter is processed, it may be contaminated when it is inserted into the circuit, so that there is a drawback that a sterile room must be provided.

[Means for Solving Problems] The present invention has been made in view of the above circumstances, and an object of the present invention is to eliminate complicated operations such as inserting a pH meter and a DO meter into a sterilized device before use. Moreover, it is another object of the present invention to provide a hollow fiber membrane cell culture device in which cells can be immediately cultured by removing the cell culture device from the sterilization device after sterilization.

And its purpose, according to the present invention, is to control at least one hollow fiber membrane cell culture unit, culture fluid reservoir, gas exchanger, pH meter, DO (liquid oxygen) meter, and perfusate flow rate. In a hollow fiber membrane cell culture device configured to include a pump, each unit constituting the culture device is made of a heat resistant material, and a hollow fiber membrane and a gas exchanger in the hollow fiber membrane cell culture unit are used. This can be achieved by a hollow fiber membrane cell culture device characterized in that high temperature and high pressure steam treatment can be performed by subjecting a porous membrane to be used to heat treatment before molding.

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

FIG. 1 is a schematic diagram showing a hollow fiber membrane cell culture device.

In the figure, the medium is introduced into the gas exchanger 4 by the pump 5 from the culture solution storage device 6, and the pH meter 1 and DO (dissolved oxygen)
The pH value and DO value of the medium during perfusion are detected by the meter 2, O ₂ and the pH adjusting gas are supplied to the perfusate in the gas exchanger 4, and the bioreactor 3 always has a constant pH value and DO value. Giving a value.

The pH meter 1 used here can withstand sterilization with high temperature and high pressure steam and maintain stable performance for a long time.
Any pH meter may be used as long as there is little fluctuation in the electromotive force of pH 7 before and after the high temperature high pressure steam sterilization operation, the sensitivity does not change, and the response speed does not slow down, especially if the pH electrode for fermentation is used. desirable.

Also, the DO meter 2 withstands repeated high-temperature high-pressure steam sterilization operations in the same manner as the pH meter, the output power before and after the sterilization operation remains stable, low residual power, and low concentration can be measured. Any DO meter may be used as long as it can respond quickly to changes in DO, and a DO electrode for fermentation is particularly desirable.

The pump 5 may be any pump as long as the liquid contact surface is completely sealed, can withstand repeated high-temperature high-pressure steam sterilization operations, and does not enter bacteria even if used for a long time. Examples thereof include a tubing pump using a tube that can withstand high-pressure steam sterilization, and a bellows pump made of a material that can withstand high-temperature high-pressure steam sterilization operation [for example, metal (SUS), heat-resistant plastic].

The culture solution storage device 6 may be any one as long as it is made of a heat resistant material, and the piping used for the circuit is also
Any material made of a heat resistant material may be used, such as stainless steel for metal, silicone and Teflon for plastic, and silicone tube is particularly desirable.

FIG. 2 is a vertical cross-sectional view showing an example of the structure of the gas exchanger used in the cell culture device. A large number of hollow fiber membranes 8 are arranged inside the tubular container 7. At both ends of the tubular container 7, the inner surface of the tubular container 7 and the outer surface of the hollow fiber membrane 8 are supported by a supporting member 9 Is kept airtight by the hollow fiber membrane 8 and both ends of the hollow fiber membrane 8 are open to both ends of the tubular container 7.

In the above-mentioned configuration, the culture solution under perfusion is the culture solution inlet 1
It enters from 0 and exits from the culture solution outlet 10 '. On the other hand, O ₂ and pH adjusting gas for the perfusate enter through the gas inlet 11, supply the gas to the culture solution through the membrane, and leave the remaining gas through the gas outlet 11 ′.

The material of the gas exchanger in the present invention may be any as long as it can withstand high-temperature high-pressure steam sterilization, for example, the cylindrical container 7 is polycarbonate, the hollow fiber 8 is polypropylene porous membrane,
Examples of the supporting member 9 include polyurethane resin,
The polypropylene porous membrane, which is a membrane for gas exchange, preferably has sufficient gas exchange ability.

In order to enable the gas exchanger to be sterilized by high-temperature high-pressure steam, it is important to subject the porous membrane used to heat treatment before molding. The heat treatment varies depending on the material of the porous film, but in the case of a thermoplastic resin, a temperature slightly lower than the melting point is preferable. For example, when polypropylene is used as the porous film, the heat treatment temperature is 120 ° C. or higher, preferably 145 to
It is carried out at 150 ° C.

When the heat treatment is usually performed for about 10 to 30 minutes at the above temperature, the shrinkage of the porous membrane is eliminated, and the porous membrane is not peeled from the support member 9 or the like during the high temperature high pressure steam sterilization treatment. Therefore, when molding is performed using a porous film that has not been heat-treated and high-temperature high-pressure steam treatment is performed, the porous film shrinks during sterilization, and the porous film peels from the support member 9 or the like. Become.

FIG. 3 is a vertical cross-sectional view showing an example of the structure of the bioreactor used in the cell culture device.

A large number of hollow fiber membranes 13 are arranged inside the tubular container 12, and the tubular container 1 is provided at both ends of the tubular container 12.
The inner surface of the hollow fiber membrane 2 and the outer surface of the hollow fiber membrane 13 are held airtight by the support member 14, and both ends of the hollow fiber membrane 13 are open to both sides of the tubular container 12.

In the above configuration, the culture solution under perfusion is the culture solution inlet 15
By entering the medium and flowing the culture solution inside the hollow fiber membrane 13, the medium is exuded to the outside of the hollow fiber membrane 13 through the pores formed in the membrane, and the cells planted outside the hollow fiber membrane 13 The culture medium is supplied to grow the cells, and the remaining culture medium is discharged from the culture medium outlet 15 '. Further, 16 is an inlet for introducing cells suspended in the culture solution into the bioreactor, the cells are planted outside the hollow fiber membrane 13, and the culture solution containing the remaining cells is discharged through the outlet 16 '. Is discharged.

The bioreactor of the present invention may be made of any material as long as it can withstand high temperature and high pressure steam sterilization. For example, the cylindrical container 12 may be polycarbonate, the hollow fiber membrane 13 may be polysulfone ultramembrane, and the supporting member 14 may be polyurethane resin.

Incidentally, the bioreactor is also similar to the above gas exchanger, by subjecting the hollow fiber membrane used to heat treatment before molding,
High temperature and high pressure steam sterilization is possible.

Therefore, next, the results of the implementation using the present cell culture device will be described more specifically.

(Example) A membrane for cell culture is a hollow fiber membrane made of polysulfone having an inner diameter of 200 μm and an outer diameter of 300 μm, an effective membrane area of 0.5 m ² , and a molecular weight cutoff of 10,000 is used, and a gas exchanger membrane has an inner diameter of 300 μm. , Outer diameter 400 μm, average pore diameter 0.2 μm,
A polypropylene hollow fiber membrane having a porosity of 65% and an effective membrane area of 0.5 m ² was used.

Further, the hollow fiber membrane for cell culture and gas exchanger,
Prior to each molding, a heat treatment operation was performed at 145 ° C. for 20 minutes.

The culture solution storage device was made of glass and 2 surge tanks (made by Hario Shibata) were used, the pump was a silicone tubing pump, and the pH and DO sensors were fermentation sensors (made by Ingold).

Connect the bioreactor, pH sensor, DO sensor, culture solution reservoir and pump with silicone tubing,
It was a closed circuit. The inside of the circuit was primed, and the entire circuit was subjected to high temperature high pressure steam sterilization at 121 ° C. for 25 minutes.

In the high-temperature high-pressure steam sterilization treatment, as described above, the entire circuit is made of a heat-resistant material, and the hollow fiber membranes for cell culture and gas exchanger are heat-treated before molding,
No peeling or damage of the hollow fiber membrane occurred.

After sterilization, the circuit was placed in a constant temperature bath at 37 ° C and serum-free Eagle's MEM-E was used as a basal medium.
(Minimum Essential Medium-Eagle) medium (penicillin potassium 100,000 units / L, kanamycin sulfate 100 mg /
(Including L) at 80 ml / min for 48 hours,
The medium was replaced with a MEM-E medium containing 10% FBS (Fetal Bovine Serum) (calf serum). 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 90 degrees every hour without circulating the medium for 4 hours to uniformly disperse the HeLa cells in the space outside the hollow fiber membrane. Then, after circulating the medium at 40 ml / min for 4 hours, the flow rate was raised to 80 ml / min, and air (1000 cc / min) and carbonic acid were added to keep the pH of the medium for cell growth at 7.4. Gas (50cc / min)
Flowed inside the gas exchanger.

The medium of No. 2 was replaced every 3 days for 15 hours, 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, the inside of the medium supply membrane is PBS (phosphate buffer solution)
(−), Replaced with 0.25% trypsin sequentially, 15 each
After culturing at 37 ° C for minutes, the cells were suspended and the cells were collected, and the number of cells was calculated. Similarly, the inside of the medium supply membrane is PBS
(−) Subsequent substitution with 2% glutaraldehyde,
After fixing with 2.5% glutaraldehyde for 24 hours,
After washing with PBS (-), it was dyed with 1% osmonic acid, freeze-dried thereafter, and gold deposition was performed, followed by SEM (scanning electron microscope) observation.

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.

From the 9th day, the glucose concentration gradually decreased, and on the 15th day, 100 mg / dl decreased to 40 mg / dl in 3 days.
Thereafter, the measurement every two days from the 16th day showed a nearly constant decrease from 100 mg / dl to 50 mg / dl.

Oxygen partial pressure and carbon dioxide partial pressure are about 150 and 20 mmHg from beginning to end.
The value of pH changes from 7.36 to 7.46.
Was stable between.

Number of cells in 31-day culture is 3 × 10 ⁸ cells / ml
Met.

As observed by SEM, the cells attached on the hollow fiber membrane were
The film entered the inside of the film and grew, and also grew outward. Cells that entered inside the membrane did not show much three-dimensional growth, but cells that grew toward the outside of the membrane were adjacent to each other and resembled the three-dimensional structure formed in vivo. Showed growth.

[Effects of the Invention] As described above, according to the hollow fiber membrane cell culture apparatus of the present invention, the porous membrane used in the bioreactor and the gas exchanger is heat-treated before being formed, and each unit of the apparatus is heat-resistant. Since it is made of a flexible material, it becomes possible to perform high temperature high pressure steam treatment on the entire hollow fiber membrane cell culture device,
The sterilization operation can be performed extremely easily.

[Brief description of drawings]

FIG. 1 is a schematic view showing a hollow fiber membrane cell culture device, FIG. 2 is a longitudinal sectional view showing an example of the structure of a gas exchanger used in the cell culture device, and FIG. 3 is a biotechnology used in the cell culture device. It is a longitudinal cross-sectional view showing an example of the structure of the reactor. 1 ... pH meter, 2 ... DO meter, 3 ... Bioreactor, 4 ... Gas exchanger, 5 ... Pump, 6 ...
Culture fluid storage device.

Claims (3)

[Claims] 1. At least one hollow fiber membrane cell culture unit, a culture medium reservoir, a gas exchanger, a pH meter, a DO (diluted oxygen) meter, and a pump for controlling the flow rate of the perfusate. In the hollow fiber membrane cell culture device configured, each unit constituting the culture device is made of a heat-resistant material, and the hollow fiber membrane in the hollow fiber membrane cell culture unit and the porous membrane used in the gas exchanger are ,
A hollow fiber membrane cell culture device characterized in that high-temperature high-pressure steam treatment is possible by performing heat treatment before molding. 2. The hollow fiber membrane cell culture device according to claim 1, wherein the porous membrane is a hollow fiber membrane. 3. The hollow fiber membrane cell culture device according to claim 2, wherein the hollow fiber membrane is made of polypropylene.