JPH0120867B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention can significantly reduce or eliminate the serum albumin concentration as well as the serum concentration in a mammalian cell culture medium. Conventionally, media for mammalian cells require the addition of mammalian serum such as fetal bovine serum or calf serum. However, these serums are generally expensive and not easily available;
Due to large lot differences, the development of alternative technologies has been considered, and it has recently been discovered that cells can proliferate at a rate comparable to that of serum media by adding human and bovine serum albumin, even without the addition of other serum components. It was done. However, since it is necessary to add a large amount of serum albumin, it is still expensive, especially when culturing cells to obtain physiologically active substances such as interferon and antibodies. The separation and purification of serum albumin is a problem, and like serum, there are considerable lot differences in serum albumin, so it is necessary to assay it in advance.
These problems were encountered with the medium containing serum albumin. As a result of various studies in order to solve these problems, the present inventors have found that by adding phospholipids, whose constituent fatty acids are unsaturated fatty acids, and mannose or galactose to the culture medium, it is possible to improve not only serum but also serum albumin. They discovered that cells could be grown at a rate comparable to that of a serum medium without the addition of almost any serum, and upon learning that this medium could solve the above-mentioned problems, they completed the present invention based on this knowledge. This is what I did. That is, the present invention relates to a medium for mammalian cells characterized by containing a phospholipid whose constituent fatty acids are unsaturated fatty acids, and mannose or galactose. The medium of the present invention must first contain phospholipids whose constituent fatty acids are unsaturated fatty acids. Phospholipids include phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidylic acid, sphingomyelin, cardiolipin, and the like. Examples of unsaturated fatty acids include oleic acid,
Mention may be made of linoleic acid, linolenic acid, arachidonic acid, erucic acid and nervonic acid. Examples of phospholipids containing unsaturated fatty acids as constituent fatty acids include phosphatidylcholine dioleoyl, phosphatidylethanolamine dioleoyl, phosphatidylcholine dioleoyl, and phosphatidylcholine monooleyl monolinoleoyl. yl, phosphatidylcholine monolinoleoyl monostearyl, and the like. It goes without saying that such phospholipids can be applied regardless of whether they are synthetically obtained or extracted from nature. Further, it does not need to be pure, and may be, for example, soybean lecithin, egg yolk lecithin, corn lecithin, cottonseed oil lecithin, rapeseed lecithin, or synthetic lecithin. If one of the constituent fatty acids of such a phospholipid is an unsaturated fatty acid, the other may be a saturated fatty acid. However, 50% or more of the constituent fatty acids of phospholipids as a whole must be unsaturated fatty acids. The appropriate content of the phospholipid in the medium of the present invention is about 0.5 to 5 μg/ml. Next, the medium of the present invention needs to contain at least one of mannose and galactose. It goes without saying that it may contain both of these two sugars. The concentration is 0.2~
Approximately 2 g/approx. Other media components include cell growth factors such as insulin and human transferrin, and nucleic acid precursors such as hypoxanthine, thymidine, deoxyadenosine, and deoxycytidine;
It is necessary to use a PH buffer such as disodium β-glycerophosphate, a sugar source such as glucose, amino acids, vitamins, inorganic salts, and other nutritional sources contained in normal serum media for cell growth. . The appropriate concentration of the cell growth factor is about 1 to 100 mg/and the concentration of the nucleic acid precursor is about 0.01 to 50 mg/. Both cell growth factors and nucleic acid precursors are used singly or in an appropriate combination of two or more, depending on the type of cells to be cultured. Glucose is usually used as the sugar source, and its concentration is preferably about 0.5 to 10 g/g/. others,
Add pyruvic acid etc. as appropriate. Amino acids are protein constituent amino acids, such as alanine,
Such as arginine, glutamine, cystine, threonine, lysine, valine, and phenylalanine. In a serum medium, essential amino acids are mainly added, but in 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. As the concentration of total amino acids
Approximately 0.5 to 5 g/approx. As for the amino acid composition, it is best to refer to a normal serum medium and supplement it with new amino acids. Vitamins include ascorbic acid, riboflavin, thiamine hydrochloride, calcium pantothenate, nicotinamide, pyridoxal hydrochloride, i-inositol,
Folic acid, VB ₁₂ , bithione, etc., and inorganic salts such as sodium chloride, potassium chloride, calcium chloride, magnesium sulfate, sodium dihydrogen phosphate, ferrous sulfate, zinc sulfate, sodium selenite, etc. can. These vitamins and inorganic salts are preferably added as appropriate to a normal serum culture medium. In addition, metabolic intermediates such as choline bitartrate, glutathione, and putrescine dihydrochloride, and buffers such as baking soda, disodium β-glycerophosphate, and N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid are added as appropriate. . Examples of basal media that should be referred to in determining the medium composition are Dulbeccos' Modified Eagle Medium, RPMI
−1640 medium, Eagle basal medium (Eaglgs'
Examples include Minimum Essential Medium), Ham F-12 medium, and the like. In conventional media, it is essential to add a large amount of mammalian serum, and in other cases, it is essential to add a large amount of mammalian serum albumin, but in the medium of the present invention, the amount of these additions can be extremely reduced. Can be done in small quantities. For example, when adding mammalian serum albumin, about 0.005 to 0.05% is sufficient. In addition, cells can be grown to a considerable extent even without adding these at all. The method for preparing the medium is to add and dissolve all the medium components to a predetermined concentration, and the order in which the medium components are added does not matter. In that case, since phospholipids have low solubility in water, it is preferable to dissolve them in ethanol or the like before adding them. After all medium components have been dissolved, the medium is sterilized by filtration, etc., and then used as a medium. Animal cells that can be cultured in the medium of the present invention are not particularly limited, and can be broadly applied to lymphoid cells, fibroblasts, epithelial cells, transformed cells thereof, cancer cells, etc. An example of such a cell is human lymphoblastoid cells transformed with Epstein-Barr Virus (EBV).
UMCL, C5180Y, human Burkitt lymphoma-derived Namalva cells, mouse lymphocyte-derived hybridoma SPI cells, human fibroblast HEL,
IMR-90, human cancer-derived epithelial cell HeLa-
S ₃ , Hep-2, KB, human primary lymphocytes, rat Yoshida sarcoma cells, hamster fibroblast BHK
-21, mouse fibroblast cell 3T3, mouse lymphoid tumor cell YAC-1, and the like. The method for ^culturing animal cells using this medium can generally be carried out in accordance with the conventional method of culturing using a serum ^medium . Add about /ml,
35-37 while venting air containing 4-6% _CO2 by volume.
It can be cultured at ℃. When culturing fibroblasts, the oxygen concentration is preferably as low as 5 to 10% by volume. It goes without saying that the medium of the present invention can be used not only for cell proliferation but also for culturing cells to produce various useful substances such as interferon, lymphokine, and antibodies. The medium of the present invention can greatly reduce the amount of serum such as fetal bovine serum, calf serum, and serum albumin, which are expensive, difficult to obtain in large quantities, and have large lot differences. Since the influence of these lot differences is small, the culture medium of the present invention is extremely suitable for mass culture of cells and mass production of useful substances such as interferon and lymphokine by cell culture. Furthermore, by reducing the amount of expensive serum and serum albumin, cells and their culture products can be produced at low cost. In particular, when the culture product is a protein, conventional media have had major problems such as separation from serum albumin and other various proteins in serum and the use of large amounts of expensive human serum albumin. There are no such problems in the culture medium. Furthermore, the medium of the present invention is particularly effective against cells that secrete glycoproteins such as interferon and antibodies. In addition, the measurement of cell number in the following examples and UMCL cell production examples is based on UMCL cells and
RPMI1788 cells were analyzed by trypan blue staining, K-562 cells by eosin staining, and Hep-2 cells by cell/cell automatic counter method. The activity of interferon is determined by the activity of human amnion-derived
Cell degeneration suppression method using FL cells and VSV (Vesicular Stomatitis Virus) (Masahiko Iizuka et al., Recent Medical Science,
29, p. 660 (1974)). In addition, immunoglobulin can be obtained after concentrating the culture solution.
Measured by diffusion sedimentation method. Example of UMCL Cell Production Approximately 20 ml of blood was aseptically collected from the umbilical cord of a newborn baby with heparin, and the lymphocytes were immediately fractionated by specific gravity centrifugation using Ficoll Isopac. This lymphocyte compartment receives 3 times the amount of
After repeating washing three times by adding Eagle MEM (Minimum Essential Medium) medium (manufactured by Nissui Pharmaceutical Co., Ltd.) and centrifuging and discarding the supernatant, RPMI containing 10 v/v% fetal bovine serum was added. 1640 medium (Nissui Pharmaceutical)
Washed lymphocytes (manufactured by Co., Ltd.) were added to a nucleated cell density of 3 x 10 ⁶ cells/ml and suspended. EBV grown in B-95-8 cells was added to this lymphocyte suspension at a concentration of 5 x 10 ⁵ TD ₅₀ /ml, and after culturing at 37°C for 2 hours, the cells were collected by centrifugation. Washing was repeated three times by adding MEM medium, centrifuging, and discarding the supernatant. Washed cells were added to RPMI containing 10v/v% fetal bovine serum.
-1640 medium at a density of 3 x ¹⁰⁶ cells/ml,
Culture was performed at 36-37°C and 5v/v% _CO2 .
Culture continued for 1.5 months, during which time 10v/v was added every 5 days.
Either an equal volume of RPMI-1640 medium containing % fetal bovine serum was added or a half volume of this medium was replaced. Three cases of umbilical cord blood were treated in this manner.
The table below shows the results of measuring the amount of spontaneous IF produced after each cell was transformed. This production amount was determined by implanting each cell into RPMI-1640 medium containing 10v/v% fetal bovine serum at a concentration of 5 x ¹⁰⁵ cells/ml under 5v/v% CO2 _and 37℃ conditions. So 5
This was obtained by measuring the activity of the culture supernatant after culturing for one day. IF activity x 10 ³ U/ml UMCL-1 2.5 Umbilical cord blood 〃 -2 6.1 Lymphocytes 〃 -3 4.5 Example 1 Sodium chloride 6400.0mg/ Potassium chloride 400.0 〃 Calcium chloride (anhydrous) 200.0 〃 Magnesium sulfate (anhydrous) 97.7 〃 Sodium dihydrogen phosphate (dihydrate)
125.0 〃 Ferric nitrate (nonahydrate) 0.1 〃 Glucose 1000.0 〃 Sodium pyruvate 110.0 〃 L-Arginine hydrochloride 84.0 〃 L-Cystine dihydrochloride 62.6 〃 Glycine 30.0 〃 L-Glutamine 584.0 〃 L- histidine hydrochloride (monohydrate) 42.0 L-isoleucine 104.8 L-leucine 104.8 L-lysine hydrochloride 146.2 L-methionine 30.0 L-phenylalanine 66.0 L-serine 42.0 L-threonine 95.2 L-tryptophan 16.0 L-tyrosine disodium 89.5 L-valine 93.6 Choline bitartrate 7.2 Folic acid 4.0 Nicotinamide 4.0mg/ Calcium pantothenate 4.0 Pyridoxal hydrochloride 4.0 Riboflavin 0.4 Thiamine salt Acid acid 4.0 i-inositol 7.2 〃 Phenol Red 5.0 〃 Dulbeccos' Modified Eagle Medium having the composition as above
The following components were added and dissolved to obtain R56-1 medium. Insulin 10mg / Human transferrin 5 Hypoxanthine 4 Thymidine 0.7 Deoxycytidine 0.03 Deoxyadenosine 1.0 6,8-dihydroxypurine 0.3 Glucose 1000 Mannose 500 Galactose 500 L -Alanine 20 〃 L-Asparagine (1 56 L-aspartic acid 20 L-cysteine hydrochloride (monohydrate) 40 L-glutamic acid 20 L-proline 20 Ascorbic acid 10 Bithion 0.2 Folinic acid 0.01 VB ₁₂ 0.1 FeSO ₄・7H ₂ O 0.8 〃 ZnSO ₄・7H ₂ O 0.02 〃 Na ₂ SeO ₃ 0.004 〃 CaCl ₂ 100 〃 Glutathione 1.0 〃 Putrescine dihydrochloride 0.1 〃 β-Glycerophosphate disodium 1500 〃 Baking soda 1300 〃 This R56− Add 0.01% human serum albumin to 1 culture medium, divide it into 9 parts, add 1/50 volume of the culture medium with an ethanol solution containing each of the following phospholipid components dissolved at 50 times the concentration, and transfer to each part using a membrane filter. It was sterilized by filtration. The table below shows each added phospholipid component and its predetermined concentration.

[Table] Ethanolamine diole oil In each culture medium prepared in this way,
UMCL-3 cells were added at 5 x ¹⁰⁵ cells/ml and incubated with air containing 5% _CO2 by volume.
The cells were cultured at ℃ for 5 days. FIG. 1 shows the results of measuring the cell concentration of the culture solution and the amount of interferon produced. In the figure, the diagonal bar graph represents the cell concentration, and the white bar graph represents the amount of interferon produced. As a control example, the right end of FIG. 1 shows a sample obtained by culturing in the same manner in which 10% by volume of fetal bovine serum (FBS) was added to RPMI-1640 medium. Example 2 Bovine serum albumin was added to the R56-1 medium prepared in the same manner as in Example 1 at a concentration of 0.05% instead of human serum albumin, divided into three, and phospholipids were added to the R56-1 medium. Zircoline dilinoleoyl (PCL) 3mg/and phosphatidylethanolamine dioleoyl (PEO)
Medium supplemented with 1 mg/ of phosphatidylcholine distearoyl (PCS) and 3 mg/of phosphatidylethanolamine distearoyl (PES)
K-562 cells were added to 2×10 ⁵ cells/ml to a medium containing 1 mg/ml of phospholipid and a medium to which no phospholipid was added (None), and CO ₂ was added to 5 ml of CO 2.
The cells were cultured at 37° C. for 5 days while aerating air containing % by volume. Figure 2 shows the results of measuring the cell concentration of the culture solution. As a control example, the right end of FIG. 2 shows the results obtained by culturing in the same manner when 10% by volume of fetal bovine serum was added to RPMI-1640 medium. Example 3 MEM medium (Minimum Essential Medium, manufactured by Nissui Pharmaceutical Co., Ltd.) 9400mg/ L-aspartic acid 13.3 L-glutamine 292 Glycine 7.5 L-glutamic acid 0.15 L-proline 3.5 L-serine 10.5 Holinine Acid 0.00005 3,3',5-triiodo-L-thyronine
0.0002 Mouse-EGF 0.01 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 ) ₆ Mo ₇ O ₂₄・H ₂ O 0.0012 〃 NiCl ₂・6H ₂ O 0.000012 〃 NH ₄ VO ₃ 0.00 0058 〃H ₂ SeO ₃ 0.00039 〃 N-2-Hydroxyethylpiperazine-N-2-
Ethanesulfonic acid 3300 〃 NaOH 300 〃 NaHCO ₃ 1400 〃 In RITC80-7 medium with the above composition
Using a medium supplemented with 0.05% human serum albumin and 10 mg/fibronectin isolated from human plasma, phospholipids were added in the same manner as in Example 2, and human pharyngeal cancer-derived cells Hep-2 were cultured. FIG. 3 shows the results of measuring the number of cells for each culture solution obtained. As a control example, the left end of FIG. 3 shows the results obtained by culturing in the same manner when 10% by volume of calf serum was added to the MEM medium. Example 4 The contents of galactose and mannose in the R56-1 medium were changed as shown in the table below, and in each case, 3 mg of phosphatidylcholine dilinoleoyl and 1 mg of phosphatidylethanolamine dioleoyl were added.
Various media were prepared by adding . Sugar concentration - 0 mg/ Glucose 1000 〃 Fucose 1000 〃 Galactose 1000 〃 Mannose 1000 〃 Galactose + Mannose 500 each 〃 UMCL-3 cells were added to each of these media in the same manner as in Example 1, and the results were Shown in Figure 4. As a control example, the right end of FIG. 4 shows the results obtained by culturing in the same manner when 10% by volume of fetal bovine serum was added to RPMI-1640 medium. Example 5 Mediums were prepared in the same manner as in Example 4, and RPMI-1788 cells were added to each medium and cultured in the same manner as in Example 1. The results are shown in FIG.

[Brief explanation of drawings]

Figure 1 shows the culture medium supplemented with various phospholipids.
This shows the results of culturing UMCL cells and measuring the cell number and interferon production amount. FIG. 2 shows the results of culturing K-562 cells and FIG. 3 Hep-2 cells in a medium supplemented with various phospholipids, and measuring the number of cells. Figures 4 and 5 show the results of examining the influence of sugar, and Figure 4 shows UMCL cells,
FIG. 5 shows the number of cells in a culture solution obtained by culturing RPMI-1788 cells in a medium supplemented with various sugars. Note that the diagonally lined bar graph in the figure represents the number of cells, and the white bar graph represents the amount of interferon or immunoglobulin produced.

Claims (1)

[Claims] 1. A medium for mammalian cells, characterized in that it contains a phospholipid whose constituent fatty acids are unsaturated fatty acids, and mannose or galactose.