JPH10108673A - Culture of animal cell using hollow yarn type incubator - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for carrying out culture at a prescribed flow rate of cultured medium in an animal cell culture by using a hollow yarn type incubator. SOLUTION: This method is to satisfy the formula, 1.5×N×(α/ c)<=(u)<=400×N×(α)/(c), wherein, flow rate of cultured medium fed to the hollow yarn type incubator is defined as (u)ml/min and live cell number of animal cells in the hollow yarn type incubator is defined as N (number), cell constant of the animal cells is defined as (α) and dissolved oxygen concentration of culture solution fed to the hollow yarn type incubator is defined as (c)g/ml.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for culturing animal cells. More specifically, the present invention relates to a method for culturing animal cells in a hollow fiber type incubator.

[0002]

2. Description of the Related Art Various methods are used for culturing animal cells, such as a stationary culture method using a petri dish or the like, a stirring culture method using a spinner flask or a tank, a roller bottle method, and the like. In addition, there is a culture method using a carrier such as a microcarrier, and improvements and developments have been actively conducted. The method of culturing animal cells using a hollow fiber type incubator uses the KAZZ method for culturing animal cells in an environment close to a living body.
Developed by EK et al. The relationship between the hollow fiber and the cultured cells in the hollow fiber type incubator simulates the relationship between a blood vessel in a living body and cells forming a three-dimensional structure around the blood vessel. In hollow fiber incubators, it is said that cells can easily maintain their original functions in vivo.In recent years, organ cells such as pancreatic cells and hepatocytes have been cultured in hollow fiber incubators to achieve the wide range of functions inherent in organs. Attempts have been made to construct hybrid artificial organs. Also, if you use a hollow fiber type incubator,
Animal cells can be cultured at a high density of 1 to 5 × 10 ⁸ cells / ml. This is because mass exchange is continuously performed between the culture solution flowing inside the hollow fiber and the cells maintained outside the hollow fiber through the wall surface of the hollow fiber, and is essential for cell growth for adherent cells. This is because a large number of cells can be secured in a limited volume as the outer surface of the hollow fiber surface. Usually, the reached density of animal cells is 10 ⁶ cells / m in stationary culture using a petri dish or a flask.
Even at l level, perfusion culture, etc., whose conditions have been well studied, 10 ⁷
Only at the individual / ml level. As described above, it is a culture method using a hollow fiber type incubator that is considered to have many advantages not found in other culture methods, but compared to other culture methods, it is actually used for industrial production of useful substances, etc. There are few examples of practical use.

In a culture method using a hollow fiber type incubator, a culture solution must be supplied to the incubator at an appropriate flow rate in order to supply nutrients to cells and remove waste products. At low flow rates, the oxygen needed to grow and maintain cells,
The supply of nutrients such as glucose and amino acids is not sufficient. In addition, cellular metabolites such as lactic acid and ammonia are not sufficiently removed, which deteriorates the state of the cells. Conversely, if the flow rate is increased too much, a situation may occur in which the state of the cells is rather deteriorated. This is because
It is thought that the cells are physically damaged by the shearing force. Serum or albumin may be added to the culture solution. Proteins in culture media have the effect of protecting cells from physical damage, but recently, in animal cell culture, they have been used as culture media to improve reproducibility, reduce cost, and produce substances for the purpose of material production. From the viewpoint of easy purification of the target product, a low serum culture solution has been frequently used. A serum-free culture or a protein-free culture may be used. As a culture method using a hollow fiber type incubator, a method using hollow fibers provided with fins (Japanese Patent Laid-Open No. 3-60477) has been developed. This improves the efficiency of supplying nutrients to the cells, but also damages the cells if the flow rate of the culture solution is inappropriate. As for the cultivation of animal cells using a hollow fiber type incubator, at present, there is no established technique that can sufficiently achieve the advantages.

[0004]

SUMMARY OF THE INVENTION An object of the present invention is to provide an effective method for culturing animal cells using a hollow fiber type incubator. More specifically, in a method of culturing animal cells using a hollow fiber type incubator, a culture method capable of long-term culturing animal cells at a high density while feeding a culture solution to a hollow fiber type incubator at a specific flow rate. The purpose is to provide.

[0005]

That is, the present invention relates to a method for culturing animal cells using a hollow fiber type incubator, wherein the flow rate of the culture solution sent to the hollow fiber type incubator is u ( ml /
Min), the number of living cells of the animal cells in the hollow fiber type incubator is N
(Unit), the cell constant of the animal cell is α, and the dissolved oxygen concentration of the culture solution sent to the hollow fiber type incubator is c (g / ml).
Where 1.5 × N × α / c ≦ u ≦ 400 × N × α
/ C. The hollow fiber used in the hollow fiber type incubator according to the present invention is a hollow fiber membrane having material permeability, and as a membrane material, usually used as a hollow fiber membrane material, polyethylene, polypropylene, cellulose, Regenerated cellulose, cellulose acetate, polysulfone, polyacrylonitrile,
Polystyrene and the like are also used in the present invention. Either a hydrophobic material or a hydrophilic material can be used. The film itself may be used, or a film coated with a substance having hydrophilicity, cell affinity, antithrombotic property, or the like, or a surface modified by chemical bonding or the like may be used.

The animal cells referred to in the present invention include both adherent and planktonic. Examples include Hela (human cervical cancer-derived cells), Balb3T3 (mouse embryo-derived cells), L929 (mouse synthetic tissue-derived cells), BGM
(African green monkey kidney-derived cells), Vero (African green monkey kidney-derived cells), BHK (newborn Syrian hamster kidney-derived cells), CHO (Chinese hamster ovary-derived cells), and cells or hybridomas into which genes have been introduced. . Not only cell lines but also primary cells such as hepatocytes and pancreatic cells, and blood-related cells such as blood stem cells, blood progenitor cells, lymphocytes, T cells, and B cells. These include cells in which a gene has been introduced into these cells, cells that have been activated by the application of an appropriate humoral factor, and the like. Further, when culturing animal cells at the culture solution flow rate according to the present invention, any means may be used as a means for sending the liquid, as long as it can send the liquid at a predetermined flow rate. It is preferable that the flow rate be variable so that the flow rate can be varied according to the state of the cells maintained in the incubator during the culture period. Examples include tube pumps, bellows type pumps, electromagnetic pumps and the like. In addition, a means for sending a liquid using gravity or pressure can also be used.

The number of viable cells in the present invention refers to the number of viable cells maintained in the incubator, but may be the number of viable cells added to the incubator at the start of culture. It also includes the number of living cells counted by the direct method and the number of living cells calculated by the indirect method. The direct method here is a method of sampling a part of the cell suspension in the hollow fiber type incubator and counting the number of viable cells contained therein under a microscope or the like. It refers to a method of indirectly calculating the number of living cells from a change in the concentration of a substance that is an index of cell metabolism, such as oxygen concentration, glucose concentration, lactate concentration, and ammonia concentration in the medium. The cell constant α in the present invention is 0.027 × 10 5 for floating cells such as hybridomas.
^-12 , 0.127x for adherent cells such as CHO
Use ^10-12 . It is a constant based on the amount of oxygen consumed by cells per unit time. The amount of oxygen consumed per unit time of animal cells to be cultured (g / cell /
If the value is known, the value may be used. The term “dissolved oxygen concentration of the culture solution sent to the incubator” as used in the present invention refers to the dissolved oxygen concentration in the culture solution immediately before being supplied to the incubator.
The dissolved oxygen concentration is determined by DO (dissolved oxyg).
en) Value. By installing a DO sensor at the culture solution inlet side of the incubator, the dissolved oxygen concentration of the culture solution at the incubator inlet side can be measured. Alternatively, a DO sensor may be provided in a bottle containing a culture solution, and if appropriate, the value may be used as the dissolved oxygen concentration of the culture solution sent to the incubator. Also, in the culture system, there is an appropriate gas exchange unit for adding oxygen to the culture solution, and if sufficient gas exchange is performed there, the dissolved oxygen concentration of the culture solution sent to the incubator An equilibrium value (for example, when culturing is performed at 37 ° C., the concentration of dissolved oxygen in pure water at 6.86 × 10 ⁻⁶ g / ml in equilibrium with the atmosphere at 37 ° C. and 1 atm) may be used. it can. As the gas exchange unit, those used for normal animal cell culture can be used in the present invention.
Examples include a gas exchanger using a silicon tube or hollow fiber, a Teflon tube wound in a coil shape and placed in a culture solution bottle, a device that blows gas directly into the culture solution in the culture solution bottle, a culture solution Examples include those that pass through the liquid surface of the culture solution in the bottle.

The present invention relates to a method for culturing animal cells using a hollow fiber type incubator at a flow rate of a culture solution of u. u
Is 1.5 × N × α / c ≦ u × 400 × N × α / c. Cells grow, maintain and proliferate by obtaining oxygen dissolved in the culture solution, but at a low flow rate less than this range, a sufficient amount of oxygen is not supplied according to the number of cells maintained in the incubator, Effective culture becomes impossible. Conversely, if the flow rate is higher than this range, the shearing force applied to the cells becomes too large, and the cells are physically damaged, and good culture results cannot be obtained. In particular, immediately after the cells are added to the incubator (at the time of addition, the cells are somewhat damaged and it is not desirable to cause further damage), or in culture using a culture solution with a low protein content, the flow rate should be increased. It is important not to raise too much. Preferably 1.5 × N × α / c ≦
u ≦ 300 × N × α / c. More preferably 1.5
× N × α / c ≦ u ≦ 150 × N × α / c. The method according to the present invention is particularly suitable for culturing animal cells using a serum-free culture solution that is susceptible to the flow rate, and particularly suitable for serum-free culturing of hybridomas for the purpose of producing antibodies.

[0009]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, description will be made with reference to embodiments.

EXAMPLE Incubators (hereinafter referred to as incubator 1) having a membrane area of 0.02 m ^{2 on the} outside of the hollow fiber and a volume of 2 ml of the outside of the hollow fiber using a hollow fiber made of regenerated cellulose, and having a membrane area of 1.
A hollow fiber type incubator (hereinafter, incubator 2) having a volume of 1 m ² and an outer volume of the hollow fiber of 60 ml was prepared. Using these incubators, a mouse-mouse hybridoma HyGPD.YK-1-1 was used.
A strain (Riken Cell No. 711) was cultured. The culture solution used Far East E-RDF (Far East Pharmaceutical).
When the cells were added to the hollow fiber type incubator at the start of the culture, the cells were suspended in a culture solution containing 10% fetal bovine serum, but thereafter, the additive RD-1 (Kyokuto Pharmaceutical) was used instead of the serum. The added serum-free culture was used.

[0010]

Example 1 A culture system comprising an incubator 1, a gas exchanger (silicon tube for gas exchange having an inner diameter of 2 mm, a film thickness of 1 mm, and a length of 2 m), a pump tube, a culture solution bottle, and a circuit tube connecting these components is prepared. did. 5 × 10 ⁶ cells were added to the incubator, and the culture was started. In this system, the concentration of dissolved oxygen in the culture solution sent to the incubator was considered to be an equilibrium value (6.86 × 10 ⁻⁶ g / ml), and the culture was started at a flow rate of the culture solution of 2 ml / min. After 2 days of culture,
A portion of the cells in the incubator were removed, stained with trypan blue, and observed under a microscope. Almost no dead cells were found, and the cells were in good condition.

[0011]

Example 2 Cells were placed in an incubator 1 in the same manner as in Example 1,
10 ⁶ cells were added, and the culture was started at a flow rate of the culture solution of 2 ml / min. The culture was continued for 64 days while the flow rate of the culture solution was kept constant. The number of viable cells at the end of the culture was 2 × 10 ⁸ . The amount of IgG produced in 64 days was determined by ELISA
It was 77.4 mg when calculated from the IgG concentration in the culture solution measured by the method.

[0012]

EXAMPLE 3 A culture system comprising an incubator 2, a gas exchanger (using a hollow fiber membrane made of silicon, a membrane area of 1.0 m ² ), a pump tube, a culture solution bottle and a circuit tube connecting these components was prepared. did. Add 5 × 10 ⁸ cells,
Culture was started. In this system, the dissolved oxygen concentration of the culture solution sent to the incubator is an equilibrium value (6.86 × 10 ⁻⁶ g / m 2).
Considering that 1), the culture was started at a flow rate of the culture solution of 50 ml / min. The flow rate was then 100 ml /
The culture was continued for 28 days at a rate of 150 ml / min from day 4 onward. The number of viable cells at the end of the culture was 2 × 10 ¹⁰ . The amount of IgG produced in 28 days was calculated from the IgG concentration in the culture solution measured by the ELISA method,
1.0 g.

[0013]

Example 4 5 × 10 ⁶ cells were added to the incubator 1
The culture was started at a flow rate of the culture solution of 6 ml / min. Two days later, some of the cells in the incubator were removed, stained with trypan blue, and observed under a microscope. As a result, some dead cells were found.

[0014]

Comparative Example 1 5 × 10 ⁶ cells were added to the incubator 1
The culture was started at a flow rate of the culture solution of 8 ml / min. Two days later, some of the cells in the incubator were removed, stained with trypan blue, and observed under a microscope. Most of the cells were dead cells.

[0015]

[Table 1]

[0016]

According to the culture method of the present invention, it is possible to appropriately supply a necessary substance to animal cells and remove waste products, and at the same time, unintended damage such as shearing force is given to the cells. Nothing. Therefore, high-density cultivation that makes full use of the advantages of the hollow fiber type incubator is possible, and can be maintained for a long period of time. Furthermore, in hybridoma culture for the purpose of antibody production,
In addition to the high-density long-term continuous culture, antibody production was improved even when a serum-free culture solution was used.

Claims (1)

[Claims] 1. A method for culturing animal cells using a hollow fiber type incubator, wherein the flow rate of a culture solution sent to the hollow fiber type incubator is u (ml / min), The number of living cells of the animal cells in the vessel is N (cells), the cell constant of the animal cells is α, and the dissolved oxygen concentration of the culture solution sent to the hollow fiber type incubator is c (g / ml). ), 1.5 × N × α / c
≦ u ≦ 400 × N × α / c.