JPS6326988B2 - - Google Patents

Description

[Detailed Description of the Invention] Fibroblasts of various mammals such as humans are known to produce useful substances such as interferon, enzymes, and polysaccharides, and these can be obtained by culturing these cells. The acquisition of useful substances has been implemented or is being considered.

For example, human fibroblasts can be cultured from human skin, human fetal organs, foreskin, etc., and recently the use of these cells as a means of producing interferon has been widely studied.

A medium supplemented with large amounts of fetal bovine serum or calf serum is usually used to culture these cells.
When performing large-scale culture, major problems were that this serum was extremely expensive and there were unexplained differences between lots. Another problem was that since bovine serum proteins were mixed in isolated and purified products such as interferon, repeated administration to humans would lead to the formation of antibodies against these proteins in the body.

The present inventors previously discovered that if mammalian serum albumin was added to the medium, it could be used as a medium for mammalian cells without adding serum; however, in the case of fibroblasts, mammalian serum albumin was added to the medium. It has been found that especially at high concentrations, cells become difficult to adhere to the culture vessel and their colonization and expansion are inhibited. In factory production, it is necessary to culture cells in a high concentration range of albumin, since the growth rate of cells is originally slow at low concentrations.

Therefore, the present inventors conducted various studies to eliminate the effect of bovine serum albumin, human serum albumin, etc. that inhibits cell colonization and spread. As a result, the present inventors determined that the oxygen concentration of the gas aerated into the culture medium was 12% or less, and that The inventors have discovered that by adding fibronectin derived from fibronectin to the culture medium, this inhibitory effect can be eliminated and the cell proliferation-promoting effect of mammalian serum albumin can be fully exerted. That is, the present invention provides that when mammalian fibroblasts are cultured in a medium containing mammalian serum albumin separated from serum, the oxygen concentration of the gas brought into contact with the medium is 3 to 12% by volume, and The present invention relates to a method for culturing mammalian fibroblasts, which comprises coating a culture container with fibronectin derived from the present invention or adding it to the culture medium.

Fibronectin is an adhesive glycoprotein that exists on the surface of mesenchymal cells and in blood, and when it is a plasma component, it is generally called cold insoluble globulin, but it is also an anti-gelatin factor. gelatin
Also called factor), microfibrillar protein, opsonic protein, etc. In the case of serum components, cell attachment factor (CAF, c-
CAP), cell spreading factor, and in the case of fibroblast surface components, fibroblast surface antigen.
antiger, SFA), galactoprotein a
It is also called galactoprotein a, cell surface protein (CSP), zeta, etc.

In the present invention, fibronectin derived from mammals such as humans, cows, horses, sheep, pigs, and mice is used. The origin of fibronectin and the origin of cells may be different; for example, fibronectin isolated from bovine serum can be used to culture human fibroblasts. Fibronectin can be produced by known methods such as gelatin affinity chromatography and column chromatography using Sepharose. The raw material is simply plasma or serum. It is desirable that the purity be 95% or higher.

To coat a culture container with fibronectin, for example, it is dissolved in a buffer solution such as phosphate buffer seline (hereinafter abbreviated as PBS), and after sterilization by filtration, this solution is coated in a culture container made of glass or plastic. You can put it in a container and leave it overnight, or you can swirl it around overnight so that the walls of the container are widely wetted, then remove it from the container, and wash the inside of the container once or several times with a buffer solution. The container may be of any type as long as its wall can adhere fibronectin to form a coating; for example, the container wall of the culture container may be coated with collagen, gelatin, procollagen, fibrin, fibrinogen, proteoglycan, etc. good. The amount of fibronectin may be about 1 to 100 mg per square meter of vessel wall area.

On the other hand, when adding fibronectin to a culture medium, it is best to dissolve it in a buffer solution such as PBS and then sterilize it by filtration before adding it. The amount added is preferably 1 mg/or more, preferably about 5 to 100 mg/. Fibronectin may be coated on glass beads, plastic beads, etc. by the method described above, and then introduced into a culture vessel. When used with coating, 2 to 5
Can be used repeatedly several times.

The medium contains mammalian serum albumin separated from serum. The feature of the present invention lies in the use of a serum-free or low serum concentration medium, and for this purpose, serum albumin is used after separating other serum components from mammalian serum.
Mammalian serum albumins include, for example, human serum albumin, bovine serum albumin, and equine serum albumin. These separations may be performed by known methods for separating serum albumin, such as ethanol fractionation, ammonium sulfate fractionation, and gel filtration. Commercially available products such as crystalline albumin and Cohn's Fraction V can be used as they are.
The appropriate amount to be added is about 0.1 to 1% by weight in the case of a serum-free medium. In the case of a medium containing serum, since serum albumin is already contained in the serum, it goes without saying that it may be necessary to have less than this.

Other medium components include cell growth factors such as insulin and human transferrin, sugar sources such as glucose, amino acids, vitamins, inorganic salts, and other nutritional sources normally included in cell growth media. A nucleic acid precursor may be added if necessary.

The appropriate concentration of the cell growth factor is 1 to 50 mg/kg, depending on the type of cells to be cultured.
Use one species or a combination of two or more as appropriate. Glucose is usually used as a sugar source at about 0.5 to 10 g/g. In addition, pyruvic acid and the like may be added as appropriate. Amino acids are protein constituent amino acids, such as alanine, arginine, glutamine, cystine, threonine, lysine, valine, and phenylalanine.

In ordinary media, essential amino acids are mainly added, but in the case of the medium of the present invention, it is preferable to add not only essential amino acids but also a wide range of non-essential amino acids. The amount added is based on the concentration of total amino acids.
Approximately 0.5 to 5 g/approx. Vitamins include ascorbic acid, riboflavin, thiamine hydrochloride, calcium pantothenate, nicotinamide, pyridoxal hydrochloride, i-inositol,
Examples include folic acid, VB ₁₂ , biotin, and inorganic salts such as sodium chloride, potassium chloride, calcium chloride, magnesium sulfate, sodium dihydrogen phosphate, ferrous sulfate, zinc sulfate, and sodium selenite. can. In addition, metabolic intermediates such as choline bitartrate, glutathione, and putrescine dihydrochloride, and buffers such as baking soda, disodium glycerophosphate, and N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid are added as appropriate.

As a method for determining the medium composition, it is preferable to use a normal cell growth medium as a basal medium, and to add serum albumin, cell growth factors, nucleic acid precursors, etc. An example of a basal medium is Dulbecco's Modified Eagle Medium.
Any known cell growth medium may be used, such as RPMI-1640 medium, Eagle's MEM medium, and Ham F-12 medium.

To prepare a medium, for example, all components except serum albumin are dissolved in water to a predetermined concentration, and then serum albumin is added and dissolved while adjusting the pH to 7.2 to 7.4 using INNaOH. The resulting solution may be sterilized by filtering it through a membrane filter under pressure.

The cells cultured in the method of the present invention are mammalian fibroblasts. Mammalian cells include humans, cows,
Cells from horses, pigs, mice, rats, etc. Examples include WI-38 cells, FS-7 cells, Vero cells, and 3T3 cells.
BHK-21 cells, and SIRC cells. The gas aerated during culture has an oxygen concentration of 3 to 12% by volume, preferably about 5 to 10%. Other components include 4 to 6% by volume of carbon dioxide gas, and the remainder is mostly nitrogen gas. As a method for preparing such a gas, for example, nitrogen gas and carbon dioxide gas in cylinders may be prepared, and these and high pressure air may be mixed to have the above composition.

Other than this, the culturing method may be carried out according to the usual method for culturing mammalian fibroblast cells. For example, put the above medium into a culture tank and add 10 ⁴ to 10 ⁶ cells.
The cells may be added at a concentration of about 100 cells/ml, and cultured at 35 to 37° C. while aerating the gas.

After completion of the culture, the proliferated cells or useful substances such as interferon can be obtained from the culture solution by conventional methods.

Using the method of the present invention, fibroblasts can be grown at a rate comparable to the conventional method using a medium supplemented with a large amount of serum, and can be subcultured over a long period of time. In addition, mouse-derived EGF (Epidermal
Growth Factor, MW6000, peptide) and 3,
3',5-triiodo-L-thyronine was required, and one problem was that these were foreign peptides and expensive, but these are no longer necessary in the culture medium of the present invention. Since serum is not added at all or in a small amount, it is easy to separate and purify the target substance, and especially when human-derived cells are cultured in a medium containing human serum albumin, all the protein components in the culture medium are removed. This method has the advantage that a target product derived from humans and produced in a culture solution can be administered to humans without having to be completely purified.

Example 1 Fibronectin obtained from human plasma was purified by gelatin affinity chromatography to obtain a product with a purity of 95% or more. Dissolve this fibronectin in a PBS solution to a concentration of 20 μg/ml.
1 ml of the solution was placed in a plastic jar (manufactured by Lux) with a diameter of 35 mm and left overnight.

The fibronectin solution was removed from the shear dish by suction and washed three times with a PBS solution to obtain a shear dish coated with fibronectin.

The medium contains R-7 medium to mouse-EGF and 3,
R- excluding 3',5-triiodo-L-thyronine
Bovine serum albumin (BSA) was added to 7E,T(-) medium at various concentrations and used. As bovine serum albumin, Cohn Fraction V manufactured by Sigma was used.
The composition of the medium is shown below.

MEM (Minimun Essential Medium, manufactured by Nissui Pharmaceutical Co., Ltd.) 9400 mg/ L-Asparagine 13.3 L-Glutamine 292 Glycine 7.5 L-Glutamic acid 0.15 L-Proline 3.5 L-Serine 10.5 Folinic acid 0.00005 human Transferrin 10 Insulin 1 VB ₁₂ 0.02 Biotin 0.02 Putrescine dihydrochloride 0.02 Sodium pyruvate 110 Choline chloride 16 Thymidine 0.07 Hypoxanthine 0.24 CuSO ₄・5H ₂ O 0.0000025 〃 FeSO ₄・7H ₂ O 0.8 〃 MnSO ₄・7H ₂ O 0.0000024 〃 (NH ₄ )Mo ₇ O ₂₄・H ₂ O 0.0012 〃 NiCl ₂・6H ₂ O 0.000012 〃 NH ₄ VO ₃ 0.000058 〃 H ₂ SeO ₃ 0.00039 〃 N-2-hydroxyethyl Piperazine-N-2
- Ethanesulfonic acid 3300 mg/ NaOH 300 〃 NaHCO ₃ 1400 〃 10% of fetal bovine serum in MEM medium (manufactured by Nissui Pharmaceutical Co., Ltd.)
HEL passaged for 15-25 generations in medium supplemented with volume%
(Human Embryonic Lung Fibroblast) cells were cultured for one generation in R-7 synthetic medium prepared by adding 0.0002 mg of 3,3',5-triiodo-L-thyronine and 0.01 mg of mouse EGF to the above-mentioned medium. The cells were incubated in a PBS solution containing 30 U/ml of crystalline trypsin for 0 to 10 minutes.
The cells were treated at 4° C. three times for dispersion, and washed three times with PBS solution to obtain a dispersed HEL cell suspension.

R-7E, 3 sheets each of the fibronectin coated and untreated sheets
Pour 1.5 ml of T(-) medium supplemented with a predetermined amount of bovine serum albumin and disperse 2 x 10 ⁴ HEL cells.
Oxygen 7% by volume prepared by mixing air, carbon dioxide gas, and nitrogen gas, sown in cells/shears.
The cells were cultured at 37° C. for 7 days while aerating a saturated humidity gas containing 5% by volume of carbon dioxide.

The culture results are shown in Figure 1. The black circles in the figure show the case where a fibronectin coated shear is used, and the white circles show the case where an untreated shear is used. The bar graph on the right side of the figure is a control.
This shows the results of culturing the same cells under the same conditions in MEM medium (manufactured by Nissui Pharmaceutical Co., Ltd.) with 10% by volume of fetal bovine serum added. In addition, the vertical axis of the figure shows the number of cells in the culture solution of each strain, and the horizontal axis shows the concentration of bovine serum albumin.

Example 2 R-7E, T(-) medium was added to three plastic bottles (manufactured by Lux) in each of the five series with a diameter of 35 mm, and the same fibronectin as in Example 1 was added at 15 μg/ml.
A medium supplemented with human serum albumin of Cone Fraction Fiber (Sigma) at a concentration of 5 mg/ml, a medium supplemented with both of these, and a MEM medium (Nissui Pharmaceutical Co., Ltd.) as a control. 1.5 ml of culture medium supplemented with 10% by volume of fetal bovine serum (manufactured by Co., Ltd.) was added to each culture medium, and human foreskin-derived cells obtained by passage in the same manner as in Example 1 were added.
Dispersed cells of FS-7 cells were seeded at 2.5 × 10 ⁴ cells/shear and cultured in the same manner as in Example 1.
Cell numbers were counted daily.

The culture results are shown in Figure 2. The vertical axis of the figure shows the number of cells in each strain, and the horizontal axis shows the number of culture days. The white circles in the figure are R-7E, T(-) medium, the black triangles are medium to which fibronectin has been added, Open triangles are medium supplemented with human serum albumin, black circles are medium supplemented with both, and open squares are MEM.
The cases are shown in which a medium containing fetal bovine serum was used.

Example 3 R-7E, T(-) medium and 15 μg/ml of the same fibronectin as in Example 1 were added to three plastic bottles (manufactured by Lux) with a diameter of 60 mm in each of three series.
and medium supplemented with bovine serum albumin at 5 mg/ml, and MEM as a control.
Example 1 The same cells as in Example 1 were seeded at 5 x 10 ⁴ cells/shear in 3 ml of each culture medium (manufactured by Nissui Pharmaceutical Co., Ltd.) supplemented with 10% by volume of fetal bovine serum. It was cultured in the same way. During culture, the two types of media used in the previous two types were dispersed at 4-5°C with a 30 U/ml crystalline trypsin solution in PBS, and the culture liquid using MEM medium was treated with a PBS solution of 30 U/ml of crystallized trypsin.
4-5 with 300U/ml crystalline trypsin in PBS
℃ treatment to disperse the cells and continue subculturing.

The culture results are shown in Figure 3. The vertical axis of the figure shows the average number of divisions, and the horizontal axis of the figure shows the number of culture days.
In addition, the white circles in the figure represent the cases in which R-7E, T(-) medium was used, the black circles in this medium with fibronectin and bovine serum albumin added, and the white squares in MEM medium supplemented with fetal bovine serum. It represents.

[Brief explanation of drawings]

Figure 1 shows the relationship between bovine serum albumin concentration and cell proliferation for vessels with and without fibronectin coating, and Figures 2 and 3 show the cell proliferation rate for various media. It is something.

Claims (1)

[Claims] 1. When culturing mammalian fibroblasts in a medium containing mammalian serum albumin separated from serum, the oxygen concentration of the gas brought into contact with the medium should be adjusted to 3 to 3.
A method for culturing mammalian fibroblasts, the method comprising coating a culture container with mammalian-derived fibronectin or adding it to the culture medium at a concentration of 12% by volume.