JP3502420B2 - Animal cell culture medium - Google Patents

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a medium for culturing animal cells.

[0002]

2. Description of the Related Art For culturing animal cells, serum, for example, fetal calf serum (FCS) is added to a commercially available basal medium in an amount of 2 to 20% as an active ingredient of the medium, and these sera are used. It involves problems such as being extremely expensive, having different lots of serum properties, and cumbersome to remove serum-derived substances from the culture product after culturing.

On the other hand, as a serum-free medium containing no serum, a method of adding protein components such as albumin, transferrin, insulin, etc. in place of the addition of serum is generally used, and such a serum-free medium is commercially available. It is available. However, the cost of transferrin, which is an essential component of this serum-free medium, is high, and a more inexpensive and effective medium component is desired.

[0004]

DISCLOSURE OF THE INVENTION The present inventors have a cell culture performance equal to or higher than that of a medium containing FCS and a serum-free medium containing transferrin as an essential component, and can be easily used industrially. We have earnestly studied the possible inexpensive cell culture medium. Specifically, without compromising the cell growth ability of the cell culture medium, FCS
In order to replace the expensive transferrin without deteriorating the cell growth ability as a serum-free medium, the study was carried out from two aspects. That is, if the amount of FCS added can be reduced, naturally there will be merit in terms of price and the amount of substances derived from serum will be reduced, so that the problems of the medium using FCS can be solved. Further, transferrin in serum-free medium is very expensive although its role and performance in cell culture medium are recognized, and it does not solve the problem in terms of FCS price. Therefore, if there is a low-cost substance that replaces transferrin, it will bring a great industrial advantage.

[0005]

Means for Solving the Problems As a result of intensive studies based on the above viewpoints, the present inventors have added a heme iron composition obtained by removing a part of the globin chain from hemoglobin by enzymatic treatment, In a cell culture medium to which FCS is added, the cell growth ability of the medium can be maintained while significantly reducing the amount of FCS to be added, and by adding a heme iron composition in place of transferrin to a serum-free medium containing no FCS, the conventional It was found that an effect equivalent to that of the FCS-added medium and the serum-free medium containing transferrin can be obtained.

The present invention is based on this finding, and is an animal cell culture medium containing a heme iron composition obtained by partially removing a globin chain from hemoglobin.

The heme iron composition used in the present invention is a hemoglobin obtained by causing an enzyme (protease) to act on hemoglobin to remove a part of the globin chain from hemoglobin. The hemoglobin is precipitated at the isoelectric point or is filtrated by ultrafiltration. The product concentrated by means such as collecting is marketed under the general name of heme iron (Hemron ^R ; Itoham Co., Ltd., Hem Ace ^R ; Asahi Kasei Kogyo Co., Ltd., Water-soluble Hemron ^R ; Ariake Food Chemical Co., Ltd., Hem Fe
ST-P ^R ; Ichimaru Farukos Co., Ltd., Hem ST-P
F ^R; Mitsubishi Kasei Foods Co., etc) is, and is administered in iron deficiency. Its properties are as follows.

The appearance is blackish brown, the iron content is 0.5 to 2.5%,
Shows high water solubility at pH 3.0 or below and 6.0 or above,
The aqueous solution is stable to heating at 121 ° C. for 30 minutes, iron exists as a divalent iron complex salt of heme-protoporphyrin, and the molecular weight is distributed in the range of 500 to 12,000, but the main active ingredient is 10 It is around 1,000.

Hemoglobin may be derived from animals other than pigs, such as cows and horses, and the iron in the complex salt may be trivalent due to oxidation.

The content of the heme iron composition is preferably 0.001-1 μg / ml, more preferably 0.005-0.1 μg / ml as an iron concentration. The heme iron composition is conveniently added to a basal medium for animal cell culture, and the basal medium is commercially available, for example, Eagle.
MEM medium, DULBECCO'S MEM medium, RP
MI 1640 medium, L 15 medium, RDP medium and the like are generally used, but are not particularly limited.

Conventionally, FCS is added to the basal medium in an amount of 2 to 20%. However, when a trace amount of the heme iron composition is added to the medium containing 10% of FCS, the colony forming rate is significantly higher than that of the medium containing only FCS. Increase (FIG. 1, Example 1).

No colony formation was observed in the medium supplemented with FCS alone at 0.1%, but the colony formation rate substantially equal to that of the medium supplemented with 10% FCS in the medium supplemented with the heme iron composition ( Example 1).

[0013] When comparing the cell growth potentials of transferrin and heme iron composition, in the serum-free medium containing no FCS, the addition of the heme iron composition was superior to transferrin in terms of colony forming rate, and 10% of FCS was added. In the medium, the heme iron composition significantly increases the viability, while transferrin does not affect the viability (Fig. 2, Example 2).

[0014]

The present invention will be further described below in the form of examples.

Example 1 A heme iron composition of the present invention or fetal bovine serum (F
The cell proliferation effect was compared between the case of adding CS), the case of not adding CS, and the case of using both in combination.

Hela cells were used as cells, and a commercially available Eagle's MEM medium was used as a basal medium. L-alanine 8.8 mg, L-asparagine monohydrate 15.0 mg, L- Aspartic acid 1
3.3 mg, L-glutamic acid 14.7 mg, L-proline 11.5 mg, L-serine 10.5 mg, glycine 7.5 mg, L-glutamine 0.292 g, dexamethasone 0.1 μM, sodium selenite 0.1 μM and The standard medium was prepared by adding 1 mg of insulin.

This standard medium or FCS to this standard medium
% And 10% added to the heme iron composition as iron concentrations of 0.005 μg / ml and 0.01 μg / m, respectively.
The following culture was carried out using a medium added so that the concentration became 1 and 0.1 μg / ml.

A plastic dish with a diameter of 60 mm containing 5 ml of each medium was trypsinized and inoculated with 500 HeLa cells washed with PBS buffer and incubated at 37 ° C. under 5% CO ₂ for 7 to 10 days. Then, methanol was added to fix the cells, and the cells were stained with Giemsa staining solution and the colonies were counted. The calculation formula for obtaining the colony formation rate is as follows.

[0019] Colony formation rate = number of colonies formed / number of inoculated cells x 100 The results are shown in Table 1 and FIG.

[0020]

[Table 1]

As is clear from Table 1, when the heme iron composition of the present invention is added to the FCS-added medium, cell growth is significantly promoted, and the heme iron composition is added to the FCS-free medium, that is, in the serum-free medium. 0.1 μg / ml as concentration
When added, a cell proliferation effect almost equal to that of FCS addition is exhibited.

Example 2 The cell growth effects of transferrin and heme iron compositions were compared, and at the same time, the presence or absence of the effect of transferrin addition on the FCS-added medium was examined.

A standard medium prepared in the same manner as in Example 1 or a medium obtained by adding 10% of FCS to each medium was added with a medium containing 0.1 μg / ml of heme iron composition as an iron concentration, and 1 μg / ml of transferrin. A medium was prepared by adding ferrous sulfate at an iron concentration of 0.1 μg / ml, and HeLa cells were cultured in these media in the same manner as in Example 1 to determine the colony formation rate. The results are shown in Table 2 and FIG.

[0024]

[Table 2]

As is clear from Table 2, the heme iron composition of the present invention shows a much stronger cell growth effect than transferrin as a serum-free medium component, and when a medium containing FCS in 10% transferrin is used. It is observed that does not affect the cell growth effect.

Example 3 The heme iron composition of the present invention and fetal bovine serum (F
The cell growth effect of the case of adding CS) and the case of adding no heme iron were comparatively examined.

Human x human hybridoma cells were used as the cells, and commercially available Eagle's M was used as the basal medium.
Using EM medium, L-arginine 8.
8 mg, L-asparagine monohydrate 15.0 mg, L
-Aspartic acid 13.3 mg, L-glutamic acid 1
4.7 mg, L-proline 11.5 mg, L-serine 1
0.5 mg, glycine 7.5 mg, L-glutamine 0.
A standard medium was prepared by adding 292 g, dexamethasone 0.1 μM, sodium selenite 0.1 μM, and insulin 1 mg.

This standard medium containing 10% FCS was used as a control, and 0.1-5% FCS was added to the standard medium.
The following culture was performed using a heme iron composition or FeSO ₄ at an iron concentration of 0.1 μg / ml and a medium to which the heme iron composition was not added.

30,000 human × human hybridoma cells were inoculated into 24-well multiplates containing 1 ml of each medium and cultured at 37 ° C. under 5% CO ₂ for 4 days.
The number of cells was counted by a Coulter counter. The results are shown in Table 3, Table 4, Table 5 and FIG.

[0030]

[Table 3]

[0031]

[Table 4]

[0032]

[Table 5]

[0033]

[Figure 3]

As is apparent from Tables 3-5 and FIG.
In the case of adding heme iron, even if the amount of FCS added is reduced to 0.5%, a cell growth effect almost equal to that of a medium containing 10% FCS is exhibited.

Example 4 The cell growth effects of the addition of the heme iron composition of the present invention and the addition of transferrin to a standard medium were compared and examined.

Human x human hybridoma cells were used as cells, and the basal medium was commercially available RPMI1640.
A standard medium was prepared by adding 0.1 μL / ml of egg yolk lipoprotein, 2.5 × 10 ⁻⁶ M sodium selenite, 0.2 mM of ethanolamine, and 2 μg / ml of insulin to the medium.

This standard medium supplemented with 10% FCS was used as a control, and the iron concentration of the heme iron composition was 0.01, 0.05, 0.1 and 0.2 μg / ml in the standard medium.
The following culture was performed using the added medium and the medium containing transferrin at 2 μg / ml.

Into a 24-well multiplate containing 1 ml of each medium, 60,000 human x human hybridoma cells were inoculated and cultured at 37 ° C under 5% CO ₂ for 4 days,
The number of cells was counted by a Coulter counter. The results are shown in Table 6 and FIG.

[0039]

[Table 6]

[0040]

[Figure 4]

[0041]

INDUSTRIAL APPLICABILITY According to the present invention, by adding a trace amount of the heme iron composition to the FCS-containing medium, it is possible to remarkably enhance the animal cell growth action, and to add serum-free medium containing no FCS to the heme iron composition. By adding the substance, the same animal cell growth effect as that of transferrin can be obtained.

[0042]

[Brief description of drawings]

1, FIG. 2, FIG. 3 and FIG.
In the graphs showing the results of the experiments in 3 and 4, the black bars in FIG. 1 represent the case where FCS of each concentration and the heme iron composition of the present invention were added to the standard medium so that the iron concentration was 0.1 μg / ml. The colony formation rate of, and the white bar indicates the colony formation rate in the case of no addition. The black bar in Fig. 2 indicates 1 FCS in the standard medium.
The colony formation rate when 0.1 μg / ml of heme iron composition was added as the iron concentration and transferrin was not added to each medium with or without 0% addition, the shaded bar indicates transferrin at 1 μg / ml and ferrous sulfate as iron. The colony formation rate when 0.1 μg / ml was added at the concentration, and the white bar shows the colony formation rate when only ferrous sulfate was added at the above concentration. The white circles in FIG. 3 are the heme iron compositions in the case where iron was not added to the standard medium, and the iron concentration was 0.1 μg / ml.
The number of proliferating cells on each culture day when added is shown. The white bar in FIG. 4 indicates the cell growth number when cultured for 4 days in a medium containing FCS at a concentration of 10% or transferrin at a concentration of 2 μg / ml in a standard medium, and the black bar indicates the iron concentration of the heme iron composition. At 0.01, 0.05, 0.1 and 0.
The number of cell proliferation when similarly cultured in a medium supplemented with 2 μg / ml is shown.

Claims (5)

(57) [Claims] 1. An animal cell culture medium containing a heme iron composition obtained by partially removing a globin chain from hemoglobin. 2. The heme iron composition has an iron concentration of 0.001.
The medium according to claim 1, which is -1 μg / ml. 3. The medium according to claim 1, wherein the medium contains fetal bovine serum and a heme iron composition. 4. The medium according to claim 1, wherein the medium contains about 0.1% or more fetal bovine serum and a heme iron composition having an iron concentration of 0.001-1 μg / ml. 5. An animal cell growth promoter in animal cell culture, comprising a heme iron composition obtained by partially removing a globin chain of hemoglobin.