JPH0739372A - Highly concentrated medium and method for cultivating animal cell - Google Patents

Abstract

PURPOSE: To obtain the subject medium enhancing cell growth and product yield and permitting the high-density culture of non-adhesive mammalian cells in vitro by balancedly fortifying all the nutrient components of a conventional cell culture medium.

CONSTITUTION: This mammalian cell culture medium for the culture of non- adhesive mammalian cells in vitro is prepared by balancedly fortifying all the nutrient components of a conventional cell culture medium, further by considering factors such as the molar concentration ratio of the components, the concentration of a cell growth inhibiting component, the ratio of mutually deeply related nutritive components, inorganic salts and the like, and the permeating of the final constituting medium. Non-adhesive mammalian cells such as a fused cell strain originated from a mouse, a human lymphoma cell or a human myeloma cell are preferably proliferated in a culture medium containing glucose in a 2.5-fold concentration and glutamine in a fivefold concentration and further containing nutritive components in fivefold concentrations, respectively, those of RPM 1-1640 culture medium.

COPYRIGHT: (C)1995,JPO

Description

Detailed Description of the Invention

[0001]

This invention relates to media traditionally used for in vitro animal cell culture, such as RPMI 164.
The present invention relates to a high-concentration nutrient-enriched medium that enables high-concentration cell culture that is close to the in-vivo cell concentration based on 0 medium, and a cell culture method using the medium. High-concentration culture medium of the present invention, in particular,
Up to now, it has been made possible to grow mammalian cells up to about 5 times the growth limit in in vitro culture.

[0002]

2. Description of the Related Art As the development of various drugs and new substances using mammalian cells has rapidly increased for the last 5 years, the most suitable process for industrialization of each product is technically and economically Immobilization technique and flow-through culture method (pe
High-concentration culture technology for mammalian cells, which is roughly classified into rfusion culture, has been rapidly introduced.

However, such a technique is based on the idea of achieving a high-concentration culture by selecting a detouring method rather than solving the complicated problems and the like of the in vitro culture method. Therefore, it was a method that had to accept various limitations (Trends Biotechnol.
5, 230, 1987 / Biotechnol. Bioeng. 28, 646, 1986 /
In vitro 22 (10), 589, 1986 / J. Immunol. Methods 8
6, 61, 1986 / Appl. Microbiol. Biotechnol. 26, 495,
1987 / European Patent No.EPO 205790,1986 / Comme
rcial Production of Monoclonal Antibodies 119, 198
6). For example, immobilization method (hollow fiber reactors cer
amic bioreactor and various immobilizing matrices
In the case of), it is difficult to increase the size of the process that is essential for industrialization due to kinetic characteristics, and in the case of a once-through culture method or a continuous culture method, it is inevitable to use an expensive medium or power. It is not easy to improve. In comparison, the cell suspension culture method has already been extensively studied for industrialization and has been used in practice, and since it does not have the problems described above, it should be reviewed in principle. Sometimes it can be said that it is relatively advantageous for industrialization (EnzymeMicrob. Techn
ol. 9, 607, 1978 / Biotechnol. Bioeng. 32, 993, 19
88).

From such a point of view, the conventional suspension culture technology has been developed by adding a specific energy source such as amino acid, vitamin, etc. to a high concentration.
This is the growth limit concentration of in vitro batch culture of animal cells (1
Although it was made to overcome ~ 3) × 10 ⁶ cells / ml, such a method showed only some effect in prolonging the survival of animal cells, which was different from the original expectation. Could not be effective in overcoming the growth limit of cells (PCT Patent WO 87 / 00195,1987 / Bio
technol. Lett. 9, 691, 1987 / WO 90/03430 (PCT / US
8903986).

The reason why such a conventional method has not succeeded in overcoming the cell growth limit is that the mutual relationship of nutrient components and the ability of cells growing in vitro to utilize nutrient components are extremely inefficient. It is not possible to recognize the facts, and in order for cells and the like to grow to a high concentration, a high-concentration culture medium in which all nutritional components and the like are fortified in a well-balanced manner is required. When viewed in principle, there is a complex interaction between the nutrient components required by a certain cell, and therefore the growth rate of the cell depends on the most exhausted component among those components. It can be said that

[0006]

However, the media and the like that have been used for cell culture so far have overcome the limiting growth concentration of in vitro cell culture and have sufficient nutrients required for the cells to grow. Had a considerably insufficient concentration of medium components (Biotechnol. Bioeng. 36, 717, 199).
0). Therefore, only high-concentration supply that reaches all nutritional components,
It can be said that such problems can be solved (Cell Culture Methods for Molecular and CellBio).
logy 1, 3, 1984).

[0007]

The present invention has developed a high-concentration medium in which a conventional medium for culturing animal cells is strengthened in a generally balanced manner, and using the newly developed medium, high-concentration cell growth is achieved. The present invention relates to a suspension cell culture method that achieves
That is, according to the present invention, for example, as shown in Table 1 below, the RPMI 1
In conventional cell culture medium such as 640 medium (J.Am. Med.Assoc.199.519.1967.), The concentration of each inorganic salt except the concentration of NaCl is left unchanged, and all amino acids, vitamins and glutathione are 5 times as much. A series of high-concentration medium groups (MBR) that have been enriched with glucose 10-fold or 50-fold and glucose 2.5-fold.
I 40-01, MBRI 40-02 and MBRI 40-
03 high-concentration medium) and the high-concentration suspension cell culturing process using the same.

In preparing the medium of the present invention, NaCl
Was adjusted from 6g / l to 4g / l, and MBRI 4
0-01 and MBRI 40-02 medium osmolality (Osmolal
ity) was adjusted to 250-320 mOsm / kg water. The MBRI40-03 medium also has an osmotic pressure (Osmolality) of 250 when the NaCl concentration is adjusted to 2 g / l or less.
It was used by adjusting it to be ~ 320 mOsm / kg in water.

Table 1 High-concentration medium group MBRI 40-01, 40-02 and 40-03, and component concentrations of RPBI 1640 medium ─────────────────────── ───────────── Culture name RPMI MBRI MBRI MBRI MBRI culture component 1640 40-01 40-02 40-03 ───────────────── ─────────────────── (Inorganic salts) (mg / l) Ca (NO ₃ ) ₂ · 100 100 100 100 100 4H ₂ O KCl 400 400 400 400 400 MgSO ₄ · _{7H 2 O 100 100 100 100 NaCl} 6000 4000 4000 - NaHCO 3 2000 2000 2000 - Na 2 HPO 4 800 800 800 800 ( other components) glutathione (reduced) 1 10 50 Phenol Red 5 5 5 5 Glucose 2000 5000 5000 5000- (Amino acids) L-Arkinin 200 1000 1000 2000 10000 L-Asparagine 50 250 500 500 2500 L-Aspartic acid 20 100 200 1000 L-Cysten 50 50 250 500 2500 L-Glutamic acid 100 200 1000 L-Glutamine 300 1500 1500-L-Glycine 10 50 100 500 L-Histidine 15 75 150 750 L-Hydroxy 20 100 200 200 1000 Proline L-Isoleucine 50 250 500 2500 L-Leucine 50 250 500 500 240 L-Lysine 200 400 2000 L-methionine 15 75 150 750 L-phenylalanine 15 75 1 50 750 L-brolin 20 100 200 1000 L-serine 30 150 300 300 1500 L-threonine 20 100 200 1000 L-tryptophan 525 50 250 250 L-tyrosine 20 100 200 1000 L-valine 20 100 100 200 1000 (vitamins) biotin 0 .20 1.00 2.00 10.0 Patent Acid Calcium 0.25 1.25 2.50 12.0 Corrin Chloride 3.00 15.00 30.00 150.0 Folic Acid 1.00 5.00 5.00 10. 00 50.0 Inositol 35.00 175.0 350.0 1750 Nicotinamide 1.00 5.0 5.00 10.00 50.0 p-Aminobenzoic acid 1.00 5.0 00.00 50.00 Pyridoxine hydrochloride 1. 00 5.00 10.0 50.0 Riboflavin 0.20 1.00 2.00 10. Thiamine hydrochloride 1.00 5.00 10.00 50.0 Vitamin B ₁₂ 0.005 0.025 0.050 0.250 ───────────────────── ───────────────

The present invention enables high-concentration culture in a high-concentration medium to give a high added value to the production of various drugs and physiologically active substances produced by animal cells. Furthermore, the present invention enables miniaturization of the conventional cell culture process, simplification and miniaturization of the purification process in order to achieve high-concentration production of the above substances, etc. It was made to be able to minimize the derived problems at the stage of conversion.

The present invention is devised so that the conventional cell culture technique does not require the use of other high-concentration culture fermenters or devices other than the use of a high-concentration medium. In addition, the burden of investment and maintenance on the device is reduced, and the size of the process can be easily increased. Matters that were taken into consideration as the main factors in the development process of the high-concentration medium are the molar concentration ratio between each component in the conventional RPMI 1640 medium, the concentration of each medium component existing in blood, and the cell growth inhibitory concentration of each nutrient component. , Factors such as the ratio between nutrient components and inorganic salts that have a close relationship with each other, the osmotic pressure of the medium, etc. (Text-Book of Medicine, 1949, 1979).

In the present invention, the result of suspension culture of a cell fusion strain 2c3.1 derived from mouse, non-adhesive mammalian cells such as human lymphoma cells and human myeloma cells using a high concentration medium , Cell concentration is about 1 × 10 ⁷ cells / ml
We were able to achieve high-concentration cultures that grow up to. Each cell line, etc. during the culture process utilizes essential nutrients such as amino acids and vitamins in a medium that is 2.5 times more concentrated in a high-concentration medium and a medium that is 5 times more concentrated.
It showed that it grew to a high concentration.

As a result of high-concentration culture, cell fusion strain 2c3.1
In this case, a single cell group antibody against the hepatitis surface anti-source was produced at a concentration 6 to 8 times higher than that which could be produced by conventional in vitro culture. In the case of fed-batch culture (fed-batch) using a high-concentration medium, the high concentration of cells is (0.7 to 1.0) × 10 ⁷ cells / ml for 2,500 hours. It was maintained for a long time, and it was possible to continuously produce a 25-fold or more single cell group antibody against the surface of hepatitis.

In order to compare the results of high-concentration culture, each cell line was inoculated at a concentration of 1 × 10 ⁵ cells / ml and 1 × 10 ⁶ cells.
MBRI 40-01 and MBRI 40-02 each containing 10% fetal bovine serum in 100 ml per cell / ml.
A high concentration medium and a conventional RPMI 1640 medium were inoculated, and at room temperature containing 5% CO ₂ at 37 ° C., 50 r
spinner culture (spinner flas)
k culture) was performed.

[0015]

EXAMPLES The present invention will be described in more detail below with reference to examples. Example 1 (High-concentration culture of cell fusion strain 2c3.1) The cultured cell fusion strain was CRL1580 myeloma cells (P3X63.A).
g. 8.653) and purified human hepatitis surface antigen (20
Balb immunized with 0 μg / ml, Green Cross Co., Ltd.
It is a cell produced by fusing pancreatic cells of c mouse, and has been deposited at the Korean Microorganism Conservation Center attached to the Korean Inoculum Association according to the Budapest Treaty (KCCM 10003).
The cells are cultured in RPM containing normal 10% fetal bovine serum.
I 1640 medium at 37 ° C. in an atmosphere containing 5% CO ₂ . The cell fusion strain serially delivers a single cell group antibody of IgGI subtype to hepatitis surface antigen,
It is a cell line that produces stably.

To compare the results of high-concentration cultures, the cell fusion strains were inoculated at 1 × 10 ⁵ cells / ml and 1 × 10 ⁶ cells / ml, each containing 10% fetal calf serum in 100 ml of MBRI. 40-01, 40-02 high-concentration medium and conventional RPMI1640 medium were inoculated and incubated at 37 ° C with 5% C
The mixture was stirred at 50 rpm in an O ₂ -containing atmosphere, and spinner flask culture was performed. The concentration of the single cell group antibody produced in the medium was quantitatively analyzed by the enzyme immunoassay as follows.

Standard single cell group antibody against hepatitis surface antigen (Anti-HBs) The cells were placed in a polystyrene well adsorbed with hepatitis surface antigen (HBsAg) and subjected to a constant temperature treatment at 37 ° C. for 2 hours. The standard antibody purified by the affinity purification method was used as a 0.15 M phosphate buffer solution (pH: 5%) containing 5% bovine serum albumin.
It diluted with 7.2) and prepared the standard solution of 4.5-300 IU / ml. After the isothermal treatment, each polystyrene well was treated with 0.1% phosphate-polysorbate-20 buffer solution 0.1.
After adding 3 ml each and washing 3 times, 100 mg of hepatitis surface antigen-horseradish perosidase conjugate diluted with 0.1 ml serum of a normal person was added and subjected to constant temperature treatment at 37 ° C. for 1 hour. . Add 0.1 ml of the substrate solution at room temperature to each well at room temperature, and after 1 hour, add 0.1 ml of the stop solution to each well.
Absorbance was measured in nm to perform a quantitative analysis of the antibody. The standard antibody used was a supernatant obtained by culturing the cell fusion strain 12c3 in a large amount and purified by an affinity purification method.

The cell fusion strain was inoculated into the conventional RPMI 1640 medium and MBRI 40-01 high concentration medium at an initial concentration of 1 × 10 ⁶ cells / ml, and the result was observed by spinner culture. As shown
In the case of RPMI 1640 medium, after 22 hours of culturing, the cells were grown to a normal cell growth limit concentration of 2.3 × 10 ⁶ cells / ml, and then the viable cell concentration was rapidly reduced. On the other hand, as a result of culturing in a high-concentration medium of MBRI 40-01, the concentration becomes about 5 times that of 9.3 × 10 ⁶ cells / ml.
It was shown to grow to a concentration of. Also, set the inoculation concentration to 1
1.5 × 10 ⁵ cells / ml which is 10 times lower than × 10 ⁶ cells / ml
When the cells were made up to ml and cultured in a high-concentration medium, they were grown to 8.0 × 10 ⁶ cells / ml which reached the inoculation concentration of 80 cultures as seen in FIG. 1, and the results according to this were obtained. Furthermore, as a result of culturing in a high-concentration medium of MBRI 40-02, the cell growth was up to 1.2 × 10 ⁷ cells /
It was grown to a concentration of ml and was shown to have about 30% more cell growth than in the MBRI 40-01 concentrated medium.

High-concentration medium MB used in this example
RI 40-02, as seen in Table 1,
BRI 40-01 medium is a medium in which all nutrients except inorganic salts are fortified twice.

The amount of single cell group antibody produced by high-concentration culture using a high-concentration medium was also 6 to 8 times higher than that produced by the conventional culture method, as seen in FIG. When the inoculation concentration of cells was 1 × 10 ⁶ cells / ml, 340 μg / ml of antibody was produced after 118 hours of culture, which was 56 μg / ml obtained when culturing in conventional RPMI1640 medium. The concentration was about 6 times higher than that of ml. Further, when the cell inoculation amount was set to about 1 × 10 ⁵ cells / ml, the antibody production amount of 450 μg / ml, which was about 8 times that in the conventional medium, was shown after 161 hours of culture. The level was slightly higher than that in the MBRI 40-01 high-concentration medium or the same level.

As a result, the high-concentration medium developed in the present invention provides a balanced supply of various nutrients and the like required for high-concentration growth above the normal growth limit concentration of the cell fusion strain. It was proved that it contributed to greatly improve the productivity of cell group antibodies.

Example 2 (Batch high-concentration culture of human lymphoma cells) Human acute lymphoblastic leukemia: CCRF-C
EM; ATCCCCL 119) is usually R containing 10% fetal calf serum
The cells were placed in PMI 1640 medium and cultured at 37 ° C. in the atmosphere containing 5% CO ₂ . To compare the results of high-concentration cultures, the cell lines were made up to an inoculum concentration (1.3-1.5) × 10 ⁶ cells / ml, each containing 10% fetal calf serum in 100 ml of MBRI 40. -01, MBRI 40-0
2 series of high concentration medium and conventional RPMI1640 medium were inoculated, the culture temperature was maintained at 37 ° C. in the atmosphere containing 5% CO ₂ , and the mixture was stirred at 50 rpm to perform spinner flask culture. did.

As shown in FIG. 3, when the cells were cultured in the conventional RPMI 1640 medium as shown in FIG. 3, after 42 hours of culture, the cell activity was increased to 3.9 × 10 ⁶ cells / ml. In contrast to the high-concentration mediums MBRI40-01 and MBRI40,
In the case of culturing at −02, after each culture for 96 hours, 8.1 × 10 ⁶ cells / ml and 8.9 × 10 ⁶ cells / m
As a result of growing at a high concentration of 1 l, not only did they grow to about twice the conventional limiting growth concentration, but
By maintaining the cell activity for 0 hours or longer, the excellent culture ability of the high concentration medium was proved.

Example 3 (batch high-concentration culture of human myeloma cells) human myeloma; RPMI 8226: ATCC CCL 1
55) is usually RPMI 16 containing 10% fetal bovine serum.
Put in 40 medium, in the atmosphere containing 5% CO ₂ at 37 ° C.
Was cultured in. The cell line was inoculated at a concentration of 1.3 × 10 ^6.
MBR containing 10% fetal calf serum as cells / ml
I 40-01, MBRI 40-02 series of high concentration medium and conventional RPMI 1640 medium 100ml each
The mixture was inoculated into the medium, and the culture temperature was maintained at 37 ° C. in the atmosphere containing 5% CO ₂ , the mixture was stirred at 50 rpm, and spinner culture was performed.

As shown in FIG. 4, when the cells were cultured in a conventional RPMI 1640 medium as shown in FIG. 4, after 28 hours of culture, the cell activity was increased to 1.5 × 10 ⁶ cells / ml. However, MBRI 40-01, which is a high-concentration medium, and MBRI 40-
In the case of culturing in 02 medium, after each culturing for 76 hours, 3.8 × 10 ⁶ cells / ml and 3.9 × 10 ⁶ cells / ml
Not only did the cells grow to a maximum of 2.5 ml and the maximum concentration of cells was increased by about 2.5 times, but the activity of the cells could be maintained for more than 50 hours.

Example 4 (Fed-batch high-concentration culture of cell fusion strain) Cell fusion strain 2c3.1 that produces single cell group antibody against hepatitis surface antigen
As in batch, concentration culture, containing 10% fetal calf serum in 100 ml at an inoculum concentration of 1 × 10 ⁶ cells / ml,
MBRI 40-02 high concentration medium was inoculated and incubated at 37 ° C for 5
In an atmosphere containing% CO ₂ , stir at 50 rpm,
Fed-batch culture was performed. Fed-batch culture is
MBRI 40-03 in Table 1 with 10% fetal bovine serum,
Glucose, glutamine (each 2.5g / 1; 750mg
Of the fed-batch culture medium to which 10% of the total culture was added so that the residual glucose concentration in the culture medium was 1 g / l.
Based on the maintenance, as shown in FIG. 5, the method of continuous addition was performed for 2,500 hours. In the figure, the arrow indicates the time of addition of the fed-batch culture medium.

As a result, as shown in FIG. 6, the cell concentration can be maintained at a high concentration of 7 × 10 ⁶ cells / ml or more and 1 × 10 ⁷ cells / ml for a minimum of 2,500 hours. As shown in FIG. 7, the amount of antibody produced is 1.0
We have developed a fed-batch high-concentration culture method that can continuously produce single cell group antibodies at g / l or higher at high concentrations for 2,000 hours or more.

As a result, the high-concentration medium developed in the present invention provides a balanced supply of various nutrients and the like required for high-concentration growth above the normal growth limit concentration of cell fusion strains. It was proved that it contributes to greatly improve the productivity of single cell group antibody.

A summary of all the above results is shown in Table 2.
Is like.

[Table 1]

As a result of batch-type or fed-batch culture of the cell fusion strain 2c3.1, human lymphoma and myeloma cells in the high-concentration medium prepared in the present invention, the growth of each cell line was the highest in the batch-type culture. The growth concentration is R, which is the control medium.
When compared with the result of culturing in PMI 1640 medium, the single cell group antibody against hepatitis surface antigen produced by the cell fusion strain was improved by 2.1 to 5.2 fold, and was 6 to 6 times higher than that in the control medium. 8 times improved. Further, when the cell line was fed-batch culture, it was possible to form a high-concentration culture in which the cell growth was improved by 3.0 to 5.2 times as much as that of the control culture for 2500 hours. It was possible to produce up to 27 times as many single cell group antibodies as compared to the expression control culture.

[Brief description of drawings]

FIG. 1 Conventional RPMI 1640 medium and RPMI
It is a growth curve comparative diagram which compared the growth of the cell fusion strain 2c3.1 cells cultivated in the high concentration medium group MBRI 40-01 and 40-02 in which all the nutrient components of 1640 medium were fortified.

FIG. 2 Conventional RPMI 1640 medium and RPMI 1
640 medium, high concentration medium M enriched with all nutrients
It is a production curve comparison figure of the single cell group antibody with respect to the hepatitis surface antigen produced by the cell fusion strain cultured in BRI40-01.

FIG. 3: Conventional RPMI 1640 medium and RPMI
It is a growth curve comparison diagram comparing the growth of human lymphoma cells cultured in high-concentration medium group MBRI40-01 in which all nutrient components of 1640 medium are fortified, and 40-02.

FIG. 4 Conventional RPMI 1640 medium and RPMI
It is a growth curve comparative diagram which compared the growth of the cultured human myeloma cell in the high concentration medium group which strengthened all the nutrient components of 1640 medium, MBRI40-01 and MBRI40-02.

FIG. 5: High-concentration medium MBRI 40-02 and 40-
FIG. 3 is a curve diagram of the residual glucose concentration during the fed-batch culturing process using No. 03 and a process diagram showing the addition method of the fed-batch high-concentration medium based on the curve diagram.

FIG. 6: High-concentration medium MBRI 40-02 and MBR
It is a cell growth curve of the cell fusion strain during a fed-batch culture process using I40-03.

FIG. 7: High-concentration medium MBRI 40-02 and MBR
It is a production curve figure of the single cell group antibody with respect to the hepatitis surface antigen produced | generated by the cell fusion strain during the fed-batch culture | cultivation process of the cell fusion strain using I40-03. that's all

[Formula 1]

─────────────────────────────────────────────────── ───

[Procedure amendment]

[Submission date] November 29, 1991

[Procedure Amendment 2]

[Document name to be amended] Statement

[Name of item to be corrected] Claim 2

[Correction method] Change

[Correction content]

[Procedure 3]

[Document name to be amended] Statement

[Correction target item name] 0004

[Correction method] Change

[Correction content]

From such a point of view, the conventional suspension culture technology has been developed by adding a specific energy source such as amino acid, vitamin, etc. to a high concentration.
This is the growth limit concentration of in vitro batch culture of animal cells (1
Although it has been attempted to overcome ~ 3) × 10 ⁶ cells / ml, such a method only showed some effect in prolonging the survival of animal cells, and the original expectation was In contrast, it could not be effective in overcoming the growth limitation of cells (PCT Patent WO87 / 0).
0195, 1987 / Biotechnol. Let
t. 9,691,1987 / WO 90/03430
(PCT / US 8903986)).

[Procedure amendment 4]

[Document name to be amended] Statement

[Correction target item name] 0007

[Correction method] Change

[Correction content]

[0007]

The present invention has developed a high-concentration medium in which a conventional medium for culturing animal cells is strengthened in a generally balanced manner, and using the newly developed medium, high-concentration cell growth is achieved. The present invention relates to a suspension cell culture method that achieves
That is, according to the present invention, for example, as shown in Table 1 below, the RPMI 1
640 medium (J. Am. Med. Assoc. 199.
519.1967. ) Etc. in a conventional cell culture medium, Na
The concentration of each inorganic salt, etc., except for the concentration of Cl, remains unchanged, and all amino acids, vitamins, and glutathione are strengthened 5 times, 10 times, or 50 times, and glucose is strengthened 2.5 times,
A series of high-concentration medium groups (MB
RI 40-01, MBRI 40-02 and MBRI
40-03 high-concentration medium) and the high-concentration suspension cell culturing process using the same.

[Procedure Amendment 5]

[Document name to be amended] Statement

[Name of item to be corrected] 0029

[Correction method] Change

[Correction content]

A summary of all the above results is shown in Table 2.
Is like.

[Table 1]

Claims (17)

[Claims] 1. For in vitro culture of non-adherent mammalian cells,
High-concentration medium enriched with all nutrients in conventional medium. 2. The composition was made in consideration of factors such as the molar concentration ratio between components, the concentration of cell growth inhibitory components, the ratio between closely related nutrient components and inorganic salts, and the permeation of the final medium of the composition. The high-concentration medium according to claim 1, which is characterized. 3. The concentration of glucose in the medium is enhanced 2.5 times and the concentration of glutamine is enhanced 5 times, and the concentration of each nutrient is
The high-concentration medium according to claim 1, wherein the high-concentration medium is fortified 5 times as much as the RPMI 1640 medium. 4. The concentration of glucose is enhanced to 2.5 times and the concentration of glutamine is enhanced to 5 times in the medium, and the concentration of each nutrient is adjusted to
The high-concentration medium according to claim 1, which is 10 times stronger than the RPMI 1640 medium. 5. The glucose concentration in the medium is 2.5.
2. The high-concentration medium according to claim 1, wherein the concentration of glutamine is enhanced by 5 times, and the concentration of each nutrient component is enhanced by 50 times that of RPMI 1640 medium. 6. The final osmotic pressure is adjusted to 25 by adjusting the concentration of sodium chloride (NaCl) without changing the concentration of other inorganic salts.
The high-concentration medium according to claim 3, 4 or 5, which is maintained in 0-320 mOs / Kg water. 7. The high-concentration medium according to claim 1, which is used for high-concentration culture of non-adherent mammalian cells of mouse-derived cell fusion strain, human lymphoma cells or human myeloma cells. 8. A non-adhesive mammalian cell derived from a mouse-derived cell fusion strain, human lymphoma cell or human myeloma cell, which has 2.5 times the glucose concentration and 5 times the glutamine concentration.
Double the concentration of each nutrient, RPMI 1640
A cell growth method, which comprises growing cells in a cell culture medium enriched to 5 times the culture medium. 9. A non-adhesive mammalian cell derived from a mouse-derived cell fusion strain, human lymphoma cell or human myeloma cell, which has a glucose concentration of 2.5 times and a glutamine concentration of 5 times.
Double the concentration of each nutrient, RPMI 1640
A cell growth method comprising growing cells in a cell culture medium which is 10 times as strong as the culture medium. 10. A non-adhesive mammalian cell derived from a mouse-derived cell fusion strain, human lymphoma cell or human myeloma cell is enriched in glucose concentration 2.5 times and glutamine concentration 5 times, RPMI 164
A cell growth method, which comprises growing cells in a cell culture medium which is 50 times stronger than 0 culture medium. 11. The cell growth method according to claim 8, wherein the cells are antibody-secreting cells. 12. The concentration of glucose is enhanced to 2.5 times and the concentration of glutamine is enhanced to 5 times, and the concentration of each nutrient is adjusted to RPM.
I A physiologically active substance produced by cells grown in a medium which is 5 times fortified with 1640 medium. 13. The concentration of glucose is enhanced to 2.5 times, the concentration of glutamine is enhanced to 5 times, and the concentration of each nutrient is adjusted to RPM.
I A physiologically active substance produced by cells grown in a medium which is 10 times fortified with 1640 medium. 14. The concentration of glucose is enhanced by 2.5 times, the concentration of glutamine is enhanced by 5 times, and the concentration of each nutrient is adjusted to RPM.
I A physiologically active substance produced by cells grown in a medium which is 50 times fortified with 1640 medium. 15. The continuous culture using the medium according to any one of claims 3 to 4 in combination for continuously producing a physiologically active substance expressed by a mammalian cell. The cell culture method according to any one of the items. 16. Addition of a combination of the culture mediums according to claims 3 to 4 to adjust the residual glucose concentration in the culture supernatant to 1 g /
The method according to claim 1, wherein the process is carried out while holding at least l.
Item 5. The cell growth method according to item 5. 17. The cell culture method according to claim 15, wherein long-term culture is performed for up to 2,500 hours.