JPH04234982A - Serum-free culture medium, method for culturing animal cell and production of foreign protein - Google Patents

Abstract

PURPOSE:To provide a serum-free culture medium, capable of exhibiting cell proliferating properties and foreign protein productivity equal to those in using a serum culture medium and suitable for growing animal cells. CONSTITUTION:A serum-free culture medium (GCM001 culture medium) is obtained by using RPMI1640 culture medium as a basal culture medium and including insulin, BP (peptone derived from beef), transferrin, HSA (human blood serum albumin), hypoxanthine, thymidine and selenium therein.

Description

[Detailed description of the invention]

0001

FIELD OF THE INVENTION The present invention relates to a serum-free medium, a method for culturing animal cells, and a method for producing heterologous proteins.

0002

[Prior art/problem to be solved by the invention] In recent years, DN
With advances in A. recombinant technology, the cultivation of animal cells for the production of cellular products (eg, proteins) has become increasingly important. To carry out such cell culture on an industrial scale, a large amount of medium is required.

[0003] Conventionally, a wide variety of media have been used to grow (proliferate) animal cells, such as DMEM medium [Morton, H. et al. J. J. (1970) In v.
itro 6,89], F12 medium [Ham, R. O.
(1965) Proc. Natl. Acad. Sci
．． USA 53,288] and RPMI 1640 medium [Goding, J. W. (1980) J. Immun
ol. Methods 39,285, JAMA 19
9 (1957) 519] etc. have been used. However, in order to grow (proliferate) animal cells using these media, serum must be added to the media. For this reason, serum from bovine fetuses, horses, humans, etc.
It had to be used at a concentration of about 15%.

However, when using a serum-containing medium, there are the following problems. ■ The cost is high because the serum itself is expensive. ■ There are lot differences in serum, which is disadvantageous for reproducible culture. ■ It becomes difficult to purify the product from the culture supernatant. ■ May become a source of virus and mycoplasma contamination.

[0005] In view of the current situation, methods of reducing the serum concentration in the culture medium are being investigated. Reducing the serum concentration in the culture medium is difficult because the decrease in serum concentration causes cells to significantly decrease their proliferative properties or die, and the yield of desired cell products (e.g., proteins) is significantly reduced. there were.

[0006] For these reasons, there is great interest in serum-free media in which cells can be cultured without losing their proliferative properties. For example, it is suitable for culturing CHO cells,
In vitro Cell. De
v. Biol. 21 (1985) 588-59
2].

However, known serum-free media have the following problems. That is, it is often necessary to add fetal bovine serum at certain time intervals during cell culture. Similarly, it is also possible to transfer basal cultures, which are normally maintained in serum-containing media, to serum-free media only by gradual adaptation.

The first object of the present invention is to provide a serum-free medium that is easy to handle, inexpensive, and suitable for the growth of animal cells.

A second object of the present invention is to provide an efficient method for culturing animal cells.

A third object of the present invention is to provide a method for producing large quantities of heterologous proteins from transformed animal cells.

[0011]

[Means for Solving the Problem] As a result of intensive research to achieve the above object, the present inventors have found that a serum-free medium containing insulin, peptone, transferrin, and albumin is suitable for culturing animal cells. The present invention was completed based on the discovery that a large amount of heterologous protein can be produced by using this medium.

That is, the first aspect of the present invention is a serum-free medium characterized in that the basal medium contains insulin, peptone, transferrin, and albumin.

[0013] The second invention is a method for culturing animal cells using the above-mentioned first invention medium.

[0014] The third invention is a method for producing a heterologous protein, which comprises culturing transformed animal cells in the above-mentioned first invention medium to express the heterologous protein.

[0015] The serum-free medium of the present invention is substantially free of serum, and usually contains a carbon source that can be assimilated by the cultured material, such as an animal cell, a nitrogen source that can be digested, an inorganic salt, etc. can be contained. Further, if necessary, trace amounts of effective substances such as trace nutrients promoting substances and precursor substances may be added. Carbon sources include glucose, mannitol, glycerol, and fructose, and nitrogen sources include organic nitrogen compounds such as meat extract, peptone, casein, corn steep liquor, soy flour, and dried yeast, and inorganic compounds such as sodium nitrate and ammonium sulfate. Examples include nitrogen compounds. The medium of the present invention uses a medium containing such components as a basal medium, and further includes the above-mentioned insulin, peptone,
This is a medium containing transferrin and albumin. As such basal medium all known media for cell culture can be used, for example DMEM
Examples of the medium include F12 medium and RPMI1640 medium, with RPMI1640 medium being preferred.

The amount of various additives in the serum-free medium of the present invention is 0.1 to 5 mg/L, especially 0.1 to 2 mg/L of insulin.
mg/L, peptone 1-10g/L, especially 4-6g/L
, transferrin 5-15 mg/L, especially 9-11 m
g/L, albumin 0.1-5 g/L, especially 0.1-2
g/L.

[0017] The insulin used in the present invention is
There is no particular restriction on its origin, and bovine origin is preferably used. The peptone used in the present invention is
There is no particular restriction on its origin, and beef-derived peptone (BP) is preferably used. The origin of the transferrin used in the present invention is not particularly limited, and those derived from humans or bovines are preferably used. The origin of albumin used in the present invention is not particularly limited, and human serum albumin (HSA) and bovine serum albumin (BSA) are preferably used.

[0018] The serum-free medium of the present invention may further contain 0.1 to 100 mg/L of hypoxanthine, especially 10 to 100 mg/L, if necessary.
15 mg/L, thymidine 0.01-100 mg/L, especially 2-5 mg/L, selenium 0.01-100 μg/L,
Among them, 2 to 5 μg/L, α-tocopherol (vitamin E
) 0.001 to 10 mg/L, especially 0.1 to 0.5 mg
/L can be added.

[0019] Furthermore, when it is desired to culture transformed animal cells containing a vector containing a resistance gene, the resistance gene contained in the vector may be added to the medium in order to maintain the stability of plasmid transformation. A corresponding selection agent may also be added. Examples of selection agents include those well known to those skilled in the art, such as neomycin, hygromycin, mycophenolic acid, hypoxanthine, xanthine, aminobutyrin or methotrexate (MTX) and derivatives thereof.

Examples of animal cells that can be cultured in the serum-free medium of the present invention include VERO, H
ela cell, chinese hamsterovar
y(CHO)Cell line, W138, BHK
, COS-7, MDCK Cell line, C12
7, HKG, Human kidney Cell li
Examples include ne. Specifically, CHO-K1 (Chinese hamster ovary cell: ATCC CCL
61), BHK (newborn hamster kidney cells: ATC
C CCL10), COS-7 (CV-1 Origi
n, SV-40 cells: ATCC CRL1651)
, Vero (African green monkey kidney cells: ATCC
CCL-81).

The method for culturing animal cells using the serum-free medium of the present invention is usually carried out as follows. That is, for culturing animal cells using the serum-free medium of the present invention, well-known culture containers such as dishes, flasks, roller bottles, spinner flasks, or culture devices using hollow fibers can be used. Not limited to these. Cultivation methods include subculturing, which is usually carried out in the culture vessels mentioned above, as well as continuous or intermittent extraction of old culture medium from the culture tank, either containing cells or separating them from the cells. Examples include, but are not particularly limited to, a continuous culture method in which cells are cultured while supplying a corresponding amount of new culture solution and controlling culture conditions constant for a long period of time.

[0022] By using this medium, adherent cells (such as CHO cells) which require a support for their growth (proliferation) can be cultured even in a suspended state without the use of a support.

[0023] The transformed animal cells used in the present invention can be prepared by means known per se.

[0024] Expression of the heterologous protein by the transformed cells may also be carried out by means known per se. Such means include, for example, the methods described in Japanese Patent Laid-Open Nos. 61-177987 and 63-146789.

According to the method for producing a heterologous protein of the present invention,
For example, antithrombin-III, prourokinase, tissue plasminogen activator, hepatitis B surface antigen, pre-S-hepatitis B surface antigen, interferon-
Heterologous proteins such as gamma, colony formation stimulating factor, etc. can be produced.

[0026]

[Examples] Examples are given below to explain the present invention in more detail, but the present invention is not limited thereto.

Example 1 ■ Preparation of serum-free medium (GCM001 medium) RPMI1640 medium [Goding, J. W(
1980) J. Immunol. Methods39,
285, JAMA 199 (1957)] 10.2g
using 1 mg of insulin, 5 g of BP (beef-derived peptone), 10 mg of transferrin, and HSA as additives.
(Human serum albumin) 1g, hypoxanthine 13mg
, 4 mg of thymidine, 0.13 mg of α-tocopherol, and 4 μg of selenium in a serum-free medium (hereinafter referred to as “GCM
001 medium. ) 1L was prepared.

■ Mutant human prourokinase-producing CHO cells (U7-2 cells) by serum-free culture (JP-A-63
Additive-dependent GCM in cell proliferation and mutant human prourokinase production (see Publication No. 146789)
U7- with the medium from which each additive was removed from the 001 medium.
2 cells were cultured, and cells in logarithmic growth phase were placed in a 6-well plate at 20 x 104 cells/well (Falcon, 3046).
in each well. The cells were cultured for 4 days at 37°C under 5% carbon dioxide, and the number of cells was counted using a Coulter counter and a hemocytometer. In addition, in order to measure the productivity of mutant human prourokinase, plasminogen activator activity in the culture supernatant was examined by a plate method.

The results are shown in FIG. A strong requirement for BP and insulin was observed for cell proliferation, and a strong requirement for insulin, transferrin, and HSA for mutant human prourokinase production.

① Examination of insulin dependence of U7-2 cells in GCM001 medium A test was conducted in the same manner as in ① above. The results are shown in FIGS. 2 and 3. Similar to the results in (■) above, it was revealed that both cell proliferation and mutant human prourokinase production were highly dependent on insulin. Furthermore, as is clear from FIG. 2, cell proliferation was dependent on the insulin concentration, and the results at 1 mg/L were the best.

■ To compare the effect of GCM001 medium on cell proliferation and mutant human prourokinase production, 10% inactivated fetal bovine serum (Bo
ehringer Mannheim GmbH, Lo
t. 698071 02) was used. In addition, ASF (manufactured by Ajinomoto Co., Inc.) is used as a serum-free medium.
The test was conducted in the same manner as in ① above.

The results are shown in FIGS. 4 and 5. It was revealed that the GCM001 medium was far superior to the serum-free medium used as a comparison in terms of both cell proliferation and production of mutant human prourokinase, and was equivalent to the serum-containing medium.

① Spinner culture using GCM001 medium In order to investigate whether GCM001 medium can be used for long-term suspension culture, spinner culture was performed using a batch method. One week after the start of culture, most of the cells in suspension were single cells, but after that, the number of clumps that appeared to be composed of several hundred cells increased. And about 1
After a few months, the majority of clamps were of approximately equal size. This clamp settled as soon as the rotation of the propeller was stopped, and by allowing it to stand still for more than 10 minutes, the medium could be replaced with a loss of about 10% of the number of cells. 2/ every 3-4 days
Three volumes of the medium were replaced, and culture continued for about 3 months. Figure 6 shows the cell number and m-PPA activity in the culture supernatant during that period. According to this, the number of cells is from 106 to 2×106
m-PPA activity was generally maintained at 100 IU/ml or more, although some fluctuations were observed.

[0034] From the above results, it was revealed that the use of GCM001 medium showed the same effect as the serum-containing medium on the proliferation of U7-2 cells and the production of mutant human prourochise. In addition, it has become possible to culture cells that originally grow in an adherent/spreading manner in a suspended state, and a suspension culture system capable of high-density culture has been established.

Example 2 ■ Mutant human prourokinase-producing DHFR activity-deficient CHO cells (Asn24-P
PA-producing cells) (see Japanese Patent Application No. 123573/1999)
cell proliferation and mutant human prourokinase-producing A
sn24-PPA producing cells are Urlaub and Ch
asin. Proc. Natl. Acad. Sci. (
USA), 77:4216 (1980) and was grown from a CHO cell line lacking DHFR activity. Asn24-PPA producing cells are cultured using the GCM001 medium obtained in Example 1, and cells in the logarithmic growth phase are implanted into each well of a 6-well plate (Falcon, 3046) at 20 x 104 cells/well. 3
The cells were cultured for 6 days at 7°C under 5% carbon dioxide, and the number of cells was measured using a Coulter counter and a hemocytometer. In addition, in order to measure the productivity of mutant human prourokinase, plasminogen activator activity in the culture supernatant was examined by a plate method.

The results are shown in FIGS. 7 and 8.

[0037]

[Effects of the Invention] By using the serum-free medium of the present invention, acclimation can be achieved in a shorter period of time than with conventional serum-free medium. It can exhibit cell proliferation and protein productivity equivalent to those obtained using a serum-containing medium, and long-term subculture is also possible. In addition, by using this medium, originally
Cells that could only be cultured by adherent/spreading growth can now be cultured in suspension.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing the cell number and plasminogen activator activity as a ratio to that in GCM001 medium.

FIG. 2 is a diagram showing the influence of insulin concentration on cell proliferation.

FIG. 3 is a diagram showing the influence of insulin concentration on mutant human prourokinase production.

FIG. 4 is a diagram showing cell proliferation of U7-2 cells in GCM001 medium.

FIG. 5 shows the production of mutant human prourokinase in U7-2 cells in GCM001 medium.

FIG. 6 is a diagram showing the cell number and m-PPA activity in spinner culture of U7-2 cells using GCM001 medium.

FIG. 7: Asn24-PPA in GCM001 medium
FIG. 2 is a diagram showing cell proliferation of production cells.

FIG. 8: Asn24-PPA in GCM001 medium
FIG. 2 is a diagram showing production of mutant human prourokinase in production cells.

Claims (3)

[Claims] 1. A serum-free medium characterized in that the basal medium contains insulin, peptone, transferrin, and albumin. 2. A method for culturing animal cells, which comprises culturing animal cells in the medium according to claim 1. 3. A method for producing a heterologous protein, which comprises culturing transformed animal cells in the serum-free medium according to claim 1 to express the heterologous protein.