JPH0239882A - Culture medium for animal cell - Google Patents

Abstract

PURPOSE:To obtain a medium having low protein content and stable performance and stably available at a low cost by adding an extraction component originated from liver to a basal medium for the culture of animal cell. CONSTITUTION:The objective culture medium for animal cell contains liver and/or liver-originated extraction component. A commercially available culture medium can be selected as a basal medium according to the kind of the animal cell to be cultured. The organ for separating the extraction component is that of cattle, pig, horse, poultry, fish, etc., and especially preferably cattle or pig organ from the viewpoint of availability. The extraction component is preferably produced by converting the organ into powder with acetone and preparing the organ-originated extraction liquid from the acetone power in use to allow the industrial mass-treatment of the extraction component and facilitate the storage of the component. The amount of the extraction component is usually adjusted preferably to give a protein concentration of 1-100mug/ml based on the basal medium.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to an animal cell culture medium, particularly to an animal cell culture medium useful for propagating animal cells such as hybridomas that produce monoclonal antibodies. .

[Conventional technology]

In general, animal cell culture media basically contain vitamins,
It is made by adding various additives to a basal medium containing inorganic salts, amino acids, etc. As for the basal medium, various compositions are commercially available, so
Although desired products can be easily obtained depending on the type of animal cells to be cultured, there are currently many uncertainties regarding the types and amounts of additives that should be added thereto.

Therefore, when evaluating the performance of a certain growth factor, the results will vary greatly depending on the individual differences of the evaluator, the type of animal cells to be cultured, the culture conditions, etc.

Under such circumstances, fetal bovine serum is known as an additive that provides relatively stable results (Reference ``Cell Culture Manual'', published by Kodansha Scientific, 1982).
). Even if serum is derived from the same cow, those derived from adult cows and calves are said to contain substances that inhibit the proliferation of animal cells, so it is generally not expected to have the same cell proliferation ability as fetal bovine serum. Can not.

However, a medium to which fetal bovine serum is added is not preferred as a medium for culturing animal cells on an industrial scale because the supply of the fetal bovine serum is not stable.

In addition, fetal bovine serum usually needs to be added at a large ratio of 5 to 15% to the basal medium, but as a result, a large amount of protein derived from fetal bovine serum is contained in the basal medium. When fetal bovine serum was added to the basal medium at 10% by volume, the protein concentration was 3000-40%.
The concentration was as high as 00 μgZml, making it extremely difficult to separate and purify useful substances produced in the culture medium from animal cells. However, reducing the amount of fetal bovine serum added is not advantageous since the rate of cell proliferation decreases.

[Problem to be solved by the invention]

In light of the above-mentioned current situation, the present invention can ensure a stable supply of protein containing low amounts and at low cost.
Moreover, it is an object of the present invention to provide an animal cell culture medium with stable performance.

[Means to solve the problem]

The animal cell culture medium of the present invention is characterized by containing extracted components derived from liver and/or stomach organs.

The present invention will be specifically explained below.

In the present invention, a medium for animal cell culture is obtained by adding and containing extracted components derived from liver and/or stomach organs to a basal medium.

In the present invention, a commercially available medium selected depending on the type of animal cells to be cultured can be used as the basal medium. As a specific example, for example, r RPMI
1640 J, "Ham F12", "Dulbetzko Modified Eagle Medium J (DMEM)", rMinimal Essential Medium J (MEM), etc. Among these, RPMl 1640, which was developed for suspension cell culture. These media can be used particularly preferably.Furthermore, these media can also be used as a mixture as appropriate.

The extracted components contained in the present invention include liver and/or kidney II! (hereinafter referred to as "specific organs"). The specific organs for obtaining this extracted ingredient are cow,
Pork, horse, poultry, fish, etc. can be used, but cow or pork is particularly preferred because they are easily available.

In the present invention, the extract component can be prepared as an extract directly from a fresh specific organ using water, phosphate buffer, about 0.15M sodium chloride aqueous solution, etc.; A method of preparing extracts derived from specific organs (hereinafter simply referred to as "extracts") from this acetone powder at the time of use enables industrial large-scale processing of extracted components and facilitates storage. It is preferable because

In the case where the extract component of a specific organ is made into acetone powder, the preparation method thereof is not particularly limited, but an example thereof is as follows.

First, connective tissue is removed from a fresh specific organ and blood is thoroughly exsanguinated, then frozen, and the frozen organ is coarsely crushed and mixed with 5 to 20 times the amount of acetone (about 20 to 10 degrees Celsius).
(hereinafter referred to as "cold acetone") and 5 to 10 times the amount of O
The mixture was poured into a blender with cold water at ~20°C, homogenized for several minutes to obtain a ground product, and the ground product was further filtered to obtain a solid.
Ether at about 10℃ (hereinafter referred to as "cold ether")
The sample is thoroughly washed with water, then dried under low temperature conditions, and if necessary, fibrous materials are removed by sieving to obtain acetone powder of the desired specific organ. This acetone powder can be stored, for example, in a cool, dark place at 5° C. or lower for a long period of time.

As mentioned above, acetone powder can be prepared using cold acetone and cold ether (Reference: New Experimental Chemistry Course 20. Biotechnology I 2゜6-7), but other organic solvents can also be used in combination. (Reference: R
Obert L, Hill et al.

Biochemical Preparation
s, pJ5. 1954>. Specific examples are as follows. For example, grind 300g of a specific organ in a frozen state with a grinder, add 100 to 200 jaws of water to a blender, and
After stirring for several minutes at about 6°C, 30 to 100 ml of an aqueous ethanol solution of about 50% by volume was added and left to stand for several hours while maintaining the temperature at about 2 to 6°C, and then 10 to 31]tl! Add 100 to 200 rnl of an approximately 95% by volume aqueous ethanol solution containing chloroform. The solid matter was separated by filtration, and this was added with 300 to 60 ml of acetone at 2 to 6°C.
0 m! ! was added and left to stand for several hours to recover the solid matter, which was again suspended in 300 to 600 d of cold acetone, and the solid matter was recovered by filtration again.
Wash sequentially with 0 mN of acetone, a mixture of 30 to 100 mN of cold acetone and cold ether, and 30 to 100 m of cold ether, then thoroughly remove the washing liquid and dry under reduced pressure to obtain the desired acetone powder. obtain.

In addition, acetone powder for specific organs is commercially available and can also be used.

The method for preparing the extract to be added to the basal medium from the acetone powder of the above-mentioned specific organ is not particularly limited, but in one example, the acetone powder is mixed with about 300 to 600 ml per 50 g of the acetone powder. Add a physiological salt solution such as IJum solution or phosphate buffer, or the same liquid as the basal medium to be used, and stir at 37°C for about 10 to 60 minutes. The suspension is centrifuged at, for example, 3000 rpm for about 10 to 60 minutes to obtain a supernatant, which is used as an extract. This extract is sterilized by filtration, and if necessary, the protein concentration is measured and stored frozen at, for example, -20°C or lower.

The protein concentration in the extract obtained as described above is usually in the range of 1 to 100 μ/-.

In the present invention, the extract obtained as described above is usually used at a protein concentration of 1 to 100% relative to the basal medium.
■ Add at a ratio of /d.

In the present invention, various substances may be added to the basal medium together with the above extract components in order to further exhibit its cell proliferation ability. As a specific example, insulin at a rate such that the concentration in the basal medium is 1 to 100 μg/mf, 1 to 100 μg/ml! Transferrin in a proportion of 2 to 200 μg/ml, ethanolamine in a proportion of 10-' to 10-9 mol/! 2-mercaptoethanol in a proportion of 1 to 100 μg/r, sodium selenite in a proportion of 10-7 to 10-9 mol/fl
Examples include fetuin with a proportion of nl.

Furthermore, in culturing using the animal cell culture medium of the present invention, it is extremely effective to add lipids to the medium.

The lipid is particularly preferably lipoprotein derived from egg yolk, and by adding this, the cell proliferation ability of the above-mentioned extract can be further enhanced. The egg yolk used to obtain lipoproteins is not particularly limited, but chicken egg yolk is preferably used from the viewpoint of ease of use and cost.

A typical method for obtaining lipoproteins from egg yolk is as follows. 0.0 for the yolk of 100 chicken eggs.
Add 1.5 to 3 fl of a 1 to 0.2 M sodium chloride solution and stir at room temperature for about 10 to 60 minutes.
Orpm for about 5 to 15 minutes to obtain a supernatant, further centrifuged at, for example, 17,000 rpm for about 10 to 60 minutes to obtain a supernatant, which is dialyzed with distilled water and further filtered and sterilized. Obtain a lipoprotein solution. The amount of this lipoprotein solution added to the basal medium is such that the protein concentration is 20 to 500 μg/mf.

Furthermore, fetal bovine serum may be added to the animal cell culture medium of the present invention.

The animal cell culture medium of the present invention includes hybridomas obtained by fusion of monoclonal antibody-producing cells and myeloma, lymphoid cells that are floating cells, hybridomas of pancreatic cells of 3alb/C mice and N5-1 cells, and black Species-derived BOWES cells, myeloma that is the parent strain used for hybridoma production, e.g. SP210-Ag-
14 (hereinafter referred to as rsP210 cells), P3/NSl
-Ag4-1 (hereinafter referred to as rNs-1 cells), etc., but is not limited thereto, and can be used to culture various animal cells.

〔Example〕

Examples of the present invention will be described below, but the present invention is not limited thereto.

Example 1 Preparation of Pig Kidney Acetone Powder Connective tissues were removed from pig kidneys immediately after slaughter, and the organ was thoroughly exsanguinated, frozen, and coarsely ground. 200g of this organ piece was mixed with 2000g of acetone kept at 4°C.
Pour into a blender with 000 g of 5℃ water and mix for 10
Homogenization was performed for a minute to obtain a ground product, which was then filtered to remove solid matter. Approximately 4500 g of this solid matter
Wash thoroughly with cold acetone, then with about 3000 g of cold ether, and dry with a blower at 5°C for 2 minutes.
Pig kidney acetone powder was obtained by removing fibrous substances from the powder by sieving. This pig kidney acetone powder could be stored in a cool, dark place at 5°C for about 6 months or more.

<Preparation of extract> 500 mj of a mixed liquid obtained by mixing liquid basal medium RPM11640 and Ham F12 at a volume ratio of 3:1!
Add to 100g of the above pig kidney acetone powder and heat at 37°C.
The resulting suspension was stirred at 300 rpm for about 30 minutes.
Centrifuge for about 30 minutes to obtain a supernatant, which is then
．． An extract was prepared by filtration and sterilization using a 22-day filter. Each of these extracts has a capacity of 50ffLl! The mixture was filled into tinips and stored frozen at -20°C. This is called “extract liquid■”
That's what it means. The protein concentration of this extract (2) was measured by spectrophotometry using Bioland protein assay and was found to be 12 mg/rnl.

Preparation of medium> 30.6 g of powdered basal medium RPMT1640 and 10.6 g of powdered basal medium Ham F12 were mixed, and this was dissolved in 3.71 g of ultrapure water. To this, 1.2 g of L-glutamic acid and 4.0 g of sodium hydrogen carbonate were added and dissolved, the pH was adjusted to 7.3 with IN aqueous sodium chloride solution, and ultrapure water was added to bring the total amount to 41. , filtered and sterilized to obtain basal medium.

The following substances were added to this basal medium to prepare a medium for animal cell culture.

Extract ■9.3ml Transferrin 56 mg Insulin 32 mg Ethanolamine 72 mg 2-mercaptoethanol 4X10-@mol Sodium selenite
6X10-@mol Example 2 Preparation of Pig Liver Acetone Powder> Fresh pig liver immediately after stratification was frozen in the same manner as in Example 1,
Pig liver acetone powder was obtained in the same manner as in Example 1, except that 300 g of coarsely crushed organ pieces, 2500 g of cold acetone, and 2300 g of 5°C water were used.

<Preparation of extract> Add 800 ml of RPM11640 medium to 120 g of the above pig liver acetone powder, stir at 37°C for about 40 minutes, and centrifuge the resulting suspension at 300 rpm for about 30 minutes to obtain a supernatant. This was filtered and sterilized using a 0.22 um filter to prepare an extract. This is called “extract liquid■”
That's what it means. When the protein concentration of this extract H was measured in the same manner as in Example 1, it was found to be 10.5/xi! Met.

Preparation of lipoprotein> Take the egg yolks from 200 fresh chicken eggs and dilute them with 0.16M chloride) Add Jum aqueous solution 4β, stir for 40 minutes at room temperature, and centrifuge this solution at 300 rpm for 10 minutes to remove the supernatant. This supernatant liquid was further heated for 17,000 r.
3. Centrifuge at pm for 30 minutes. After obtaining the OR supernatant and dialyzing it with distilled water,
A lipoprotein solution was prepared by filtration and sterilization in this order through JLQl and 0.22u+n filters, which was divided into small vials and stored at -20°C. This is called "lipoprotein solution A." The protein concentration in this lipoprotein solution was measured in the same manner as in Example 1 and was found to be 19 mg/-.

<Preparation of medium> 5. The following substances were added to OA's liquid basal medium RPM11640 to prepare a medium for animal cell culture.

Extract II 9rnl lipoprotein solution A 22-transferrin
85 ■Insulin
55 ■Ethanolamine 90
mg2-Mercaptoethanol 6 x 10-mol Sodium selenite 5 x 10-mol Example 3 Preparation of beef kidney acetone powder> Immediately after stratification, cow kidney was treated in the same manner as in Example 1, and 350 g of coarsely ground organ pieces were obtained. Beef organ acetone powder was obtained in the same manner as in Example 1 except that the ground product was washed with 5200 g of cold acetone using 3300 g of cold acetone and 3400 g of cold water.

Preparation of extract> Liquid basal medium RPM11640, Nome F12 and DMI
EM and EM at a volume ratio of 3:1:1 was added to 90 g of the acetone powder and stirred for about 40 minutes at 37°C.
Centrifuge at °rpm for about 30 minutes to obtain a supernatant.
This was filtered through a skin 22 filter and sterilized to prepare an extract. This is called "extract liquid ■." When the protein concentration of this extract (■) was measured in the same manner as in Example 1, it was found to be 13.7 mg/mf! Met.

Preparation of culture medium> Mix 30.6 g of powdered basal medium RPMi1640, 10.6 g of powdered basal medium RPMi1640 and 9.5 g of OIAEM, dissolve this mixture in ultrapure water of 4.61, and add to this. Add and dissolve 1.5 g of L-glutamic acid and 5.0 g of sodium bicarbonate, adjust the pH to 7.3 with an aqueous IJ solution, and further add ultrapure water to bring the total amount to 5.0 g. After making it into Ol, it was filtered and sterilized to obtain a basal medium. The following substances were added to this basal medium to prepare an animal cell culture medium.

Extract (III) 9 mj!
Lipoprotein Solution A 16.4 dTransferrin 43 mg Insulin 63 mg Ethanolamine 110 mg 2-Mercaptoethanol 6X10-@mol Sodium Selenite
6X10-”Morphetwin 7
2 mg Example 4 <Preparation of extract> In Example 1, instead of pig kidney acetone powder, porcine stomach acetone powder rK3875' (manufactured by Nguma, USA) 1
An extract was obtained in the same manner as in Example 1, except that 50 g of ltPM(1640 and )\muF12 were used and 600 ml of phosphate buffer was added instead of the mixed solution of ltPM(1640 and )\muF12. This is called “extract liquid■
”. The protein concentration of this extract (2) was measured in the same manner as in Example 1 and was found to be 17 mg/m.

<Preparation of medium> A medium for animal cell culture was prepared in the same manner as in Example 3 except that extract IV7.3d was used instead of extract I[[9mf.

Example 5 Preparation of culture medium> In Example 3, the amount of extract liquid ■ added was 23.6 ml! An animal cell culture medium was prepared in the same manner as in Example 3, except for binding.

Example 6 <Preparation of medium> In Example 3, the amount of extract ■ added was 20.7 ml! An animal cell culture medium was prepared in the same manner as in Example 3, except that the amount of lipoprotein solution A added was 22.7 rnl.

Experimental Example Culture Test 1 Established cell line derived from mouse myeloma 5
Animal cell culture medium prepared in Examples 1 to 6 using P210 cells, N5-1 cells, a hybridoma of Ba1b/C7 mouse pancreatic cells and N5-1 cells, and 80WES cells derived from the black variety as cultured cells. A culture test was conducted using the following procedure.

1 micrometer of the animal cell culture medium prepared in Examples 1 to 6 was placed in each well of a 24-well multi-well for cell culture, and equilibrated at a temperature of 37°C under a 5% carbon dioxide atmosphere. The above cells were seeded at a ratio of lXl0' each to make six sets per cell type, and the temperature was 3.
The cells were cultured at 7°C under a 5% carbon dioxide atmosphere. On the 3rd and 5th day after the start of culture, the number of cells was counted using a Coulter counter, and the average number of cells in three wells under the same conditions was determined.

In addition, similar culture tests were conducted using the following four types of media as control media.

Control medium 1: RPMI 1640 Control medium 2: 3RDF (basal medium RPM11640
, I]Medium containing 3:1:1 mixture of RDF and Ham's F12) Control medium 3: RPM11640 with 0.3% by volume beef fat serum Control medium 4: 3RDF with 0.3% by volume beef tallow The protein concentrations of control mediums 3 and 4 to which infant serum was added are approximately the same as those of extract solution ①.

The results are shown in Tables 1 to 4.

Table 2 (NS-1 cell culture test) Table 1 (SP210 cell culture test) Table 3 (Hybridoma culture test) Table 4 (BOWES cell culture test) The raw materials are also liver and/or kidney. Therefore, when using animal cells to industrially produce monoclonal antibodies and useful physiologically active substances, it is possible to ensure a low-cost and stable supply of animal cells. It can also be advantageously used for culture.

〔Effect of the invention〕

Claims (2)

[Claims] (1) An animal cell culture medium characterized by containing an extracted component derived from liver and/or kidney. (2) The animal cell culture medium according to claim 1, which contains lipoprotein derived from egg yolk.