JPS637780A - Feeding method for iron to cell and serum-free synthetic culture medium used thereof - Google Patents

Abstract

PURPOSE:To make it possible to carry out multiplication and subculture of cells in a serum-free synthetic culture medium, by forming a stable complex of iron with a chelating agent and containing the resultant complex in the serum-free synthetic culture medium. CONSTITUTION:A chelating agent, e.g. ethylenediamine dipropionate hydrochloride, hydroxyethyliminodiacetic acid, iminodiacetic acid, etc., capable of forming at least a stabler complex with trivalent iron ions than bivalent iron ions under neutral or alkaline condition, preferably having <=15 chelate stability constant with the trivalent iron ions and the trivalent and/or bivalent iron ions are contained in a serum-free synthetic culture medium prepared by adding a small amount of transferrin, as desired, thereto and the iron is converted into the stable complex with the chelating agent and fed to cells.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a method for stabilizing iron ions with a chelating agent and supplying the same to cells, and a serum-free synthetic medium used therein.

[Prior Art] For example, in order to separate animal cells from a living body and culture them in a so-called test tube, the cells are treated with an isotonic buffer solution containing carefully selected nutrients (amino acids, vitamins, sugars, electrolytes, etc.). media - these are Eagle's) IB
A culture solution prepared by adding an appropriate amount of animal serum to a well-known medium such as M.M., RPM11640, and Ham's F12 medium is required.

At this time, serum not only plays the role of creating a nutritional environment necessary for cell growth and proliferation, but also plays an important role in intracellular replenishment of various cell growth factors, fat-soluble vitamins, lipid components, and various metal elements. is in charge of the mission. In other words, the presence of serum made it possible to culture cells for the first time.

However, it is a well-known fact that the quality of serum varies greatly depending on the animal's breeding environment, type of feed, health condition, time of blood collection, and place of production. This allows us to obtain high quality serum. Although it takes time and effort to select these sera, the suitability of the sera is directly linked to the success or failure of the culture, and the serum is so expensive that it accounts for over 80% of the cost of the culture solution, so it cannot be neglected.

In this way, even with serum-supplemented media that requires time and effort, the presence of serum components becomes an obstacle when extracting and purifying industrially or medically important active factors from cultured cells. Removal operations are required.

Therefore, there has long been a demand for the development of a so-called serum-free medium, which is composed only of chemically synthesized components without adding serum.

However, the reality is that the serum-free media published so far contain serum albumin fractions whose constituent components have not been chemically elucidated, or contain transferrin protein separated from serum as an essential factor. It is.

Representative examples of serum-free media that have been published so far are shown below for reference.

(D CITTL medium [Dulflair (F, J,
Darf'1er), Incel (P, A, In5el
) et al., 1982] Basal medium - Dulbecco's modified Eagle medium 10 μm F12 medium (1:1) Additives - casein, insulin, testosterone, linoleic acid (n) 5FFD medium (H, Murakami et al., 1982) ) Basal medium - Dulbecco's modified Eagle medium 10 μm F
12 medium (1:1) Additives - insulin, transferrin, ethanolamine, selenium (to) KSLM medium (T, Kawamoto et al., 1983)
Basal medium - PPM11640 + Dulbecco's modified Eagle's medium + Ham's PL2 medium (2:1:1) Additives - insulin, transferrin, low-density lipoprotein, free fatty acid-free bovine serum albumin, oleic acid, ethanolamine, 2-mercapto As mentioned above, the performance of a medium supplemented with ethanol, selenium, and Hepes serum is affected by the quality of the serum, but when albumin is added to a serum-free medium, contaminant components contained in albumin (albumin is (It is said that it is functionally combined with many important elements.) has a considerable influence on the performance of the culture medium.

Furthermore, even when serum or serum albumin, which causes fluctuations in medium performance, is removed, transferrin is still required as a trace addition factor to serum-free medium. Transferrin binds to iron, an essential element for cells,
It has been proven that it plays a role in being taken into cells, and it has continued to be used in serum-free media despite being an expensive serum protein component.

[Problems to be solved by the invention] Since the supply of iron is essential in the culture of animal cells, conventionally albumin fractions and expensive transferrin were added to synthetic media as serum or serum components. Therefore, the cost of the culture solution has increased or the quantity has been limited.

Furthermore, when extracting a target component from a culture solution, these added proteins become impurities and require a troublesome removal operation.

For example, transferrin added to conventional serum-free media is usually industrially separated and purified from human serum and is expensive, with a concentration of approximately 5 to 100 ml per liter of culture solution.
g, and accounts for about 20-40% of the culture solution cost. When considering substance production using animal cells, replacing part or all of the transferrin protein with an inexpensive chemically synthesized substance has great advantages not only in reducing the cost of the culture solution but also in purifying the product.

In addition, it is said that the fewer protein components there are in the culture of cells used for vaccine virus propagation, the fewer side effects (such as anaphylaxis) during vaccination will occur, so it also greatly contributes to safety in vaccine production.

Furthermore, in research fields related to cell culture such as medicine, biology, and agriculture, the theme that is currently attracting the most attention is the analysis of the mechanisms of canceration and aging in living organisms. In addition to conventional media that require serum, serum components such as albumin and transferrin are considered to contain considerable anti-cancer and aging factors, so it is important to provide a culture system that does not contain serum components. The contribution is significant as it promises to advance new research.

Furthermore, from a clinical medical perspective, it will be useful in elucidating the pathogenesis of anemia and metal element deficiency diseases caused by gastric ulcers, etc. or genetic predisposition, and will also provide new methodologies for treatment.

On the other hand, it has been experimentally proven that iron uptake into cells without transferrin is possible by rapidly bringing divalent iron into cells.

However, divalent iron is immediately oxidized in the liquid state (neutral to weakly alkaline) of commonly used animal cell culture media and precipitates as ferric hydroxide, which is not industrially practical.

The present invention enables cell culture in a serum-free medium that does not contain transferrin by formulating iron as more stable trivalent iron and at the same time adding a chemical compound that appropriately chelates iron. It is intended to replace part or all of the protein component transferrin.

[Means for Solving the Problems] That is, an object of the present invention is to supply the easily insolubilized iron element required by cells into cells in a stable form.

BACKGROUND ART Conventionally, a method is known in which a chelating agent is administered for the purpose of detoxifying toxic heavy metals that have been accidentally ingested, and a stable complex is formed in the body to be excreted. In other words, such a chelating agent has the effect of insolubilizing the internal organs and tissues of the body, solubilizing the deposited heavy metals, and removing them from the body.

The present inventors grasped the significance of the detoxification effect of such chelating agents, and as a result of intensive research, it was found that certain chelating agents have positive effects on cells by stabilizing metal elements that tend to form precipitates as complexes. This was the first time in the field of cell culture that it was established that it could be supplied to the human body.

Specifically, the present invention provides a method for supplying iron to cells, which is characterized by supplying iron to cells after being stabilized with a chelating agent, and a serum-free synthetic medium for cells, which is characterized by containing iron ions and a chelating agent. This applies to

Here, the iron taken into cells is mainly trivalent iron ions, but cells can also take in divalent iron ions.

Therefore, inorganic or organic ferric salts or inorganic or organic ferrous salts can be used as the iron source. Any of these iron sources can be used as long as it does not adversely affect the cultured cells, but soluble ones such as ferric chloride and ferrous sulfate are preferred.

However, since the PL of the culture solution or body fluid is neutral or slightly alkaline, divalent iron ions are easily oxidized to trivalent iron ions, which further become ferric hydroxide and precipitate.

The chelating agent used is one that is neutral to weakly alkaline and forms a stable complex with trivalent iron ions.

Those that form a more stable complex with trivalent iron ions than with divalent iron ions are preferred. However, if the binding force with trivalent iron is too strong, it will be an obstacle to supplying trivalent iron ions to cells, so the chelate stability constant (1og K
ML) is 15 or less. Examples include ethylenediamine nipropionic acid hydrochloride, hydroxyethyliminodiacetic acid, iminodiacetic acid, and the like.

The cells to which iron is supplied may be of animal or plant origin, and the present invention is applicable to all iron-dependent cells.

Various formulations of the medium can be used, and for example, the formulations shown in Examples below are suitable. Moreover, a commercially available basal medium can also be used.

As the basal medium, general serum-free synthetic media such as Eagle's HEM, RPMI 1640, Dulbecco's modified Eagle's medium, Ham's F12, etc. can be used alone or in combination of two or more.

In the above, the concentration of iron ions per culture solution 11 is 0,
05~loaM is appropriate, and the concentration of the chelating agent is 0.05~loaM.
1-15mM is suitable.

Essentially, there is no need to add transferrin, albumin fraction, serum, etc., but a small amount of transferrin is required (
It may be added accordingly.

Next, a method for preparing the serum-free synthetic medium of the present invention will be described.

First, a double concentration serum-free synthetic medium (containing growth factors such as insulin, ethanolamine, selenium, epidermal growth factor, triiodothyronine, etc.) is prepared in advance. Separately, a chelating agent solution of 0.2 to 30 mM is prepared, and an iron compound solution is also prepared, and the iron compound solution is first added to the chelating agent solution to form a complex. Thereafter, equal amounts of the double concentration serum-free synthetic medium and the complex solution are mixed.

It is important to bind the metals and the chelating agent in advance; if they are mixed alone with other components of the medium without this pretreatment, the effectiveness will be halved.

The preparation method will be shown using iron as an example. 0.2 to 2 g of an inorganic salt of divalent iron or trivalent iron is dissolved in 0.01 N hydrochloric acid to give a concentration of 11, and 1 volume of the solution is added to 99 volumes of the chelating agent solution. The complex solution thus prepared is mixed in equal amounts with double concentration serum-free synthetic medium to obtain the desired transferrin-free culture solution. Alternatively, transferrin may be supplemented as needed. Alternatively, it is of course possible to prepare a complex solution in advance and sequentially add other medium components to it to formulate a desired serum-free synthetic medium.

Of course, the finally obtained culture solution is temporarily sterilized and stored aseptically.

Insulin, selenium, etalamine, epidermal growth factor, triiodothyronine, etc. that are supplemented to the synthetic medium are already known substances that contribute to improving cell proliferation, and are serum-free. It forms part of the essential components in culture.

[Effect] Using the thus prepared serum-free synthetic medium that does not contain albumin and transferrin or contains a trace amount of transferrin, lymphoid cells such as No. 1 hybridoma and interstitial cells such as fibroblasts can be cultured. Alternatively, when epithelial cells such as kidney cells were cultured, good culture results were obtained as shown in the following test examples.

Test Example 1 Mouse hybridoma C2a''-E was grown for 2 days in a medium to which no serum or chelating agent was added (for example, a 1:1 mixture of Eagle MEN and RPM11840 medium).
After pre-culturing T cells with 5% carbon dioxide gas in a Cape Oven at 37°C and arresting the cell cycle, T cells were incubated with chloride based on each culture medium and the formulation of Example 1.2.3 described below. 11 each for the medium with diiron removed and transferrin added and the medium without chelating agent as a control! Dispense the same medium into 3 wells of a 12-well culture plate and pre-culture C2a-E.
5 x 10' cells of each cell were seeded and main culture was carried out for 3 days in a 5% carbon dioxide egg disintegrator at 37°C. The ability of each medium to support cell proliferation was determined by counting the number of viable cells that had proliferated. The results are shown in Table-1.

It is clear that the use of chelating agents to replace the intracellular supply of iron by transferrin, which is normally provided by the addition of serum, allows cell proliferation. However, supplementation of iron alone without the addition of transferrin or chelating agents does not result in cell cycle recirculation.

This demonstrates the effectiveness of the compound used as a chelating agent.

Test Example 2 The medium of Example 1 described later, a medium from which ferric chloride has been removed from this formulation, or apotransferrin that does not bind iron or iron ions to these iron-added or iron-free medium is saturated. Prepare a medium supplemented with holotransferrin, etc., or a serum-free medium that does not contain a chelating agent, and test the ability to support the growth of hybridoma (C2a-El) in each of these medium according to the procedure of Test Example 1. It was determined by The results are shown in FIG.

In the presence of free iron or holotransferrin, chelating agents significantly improve cell proliferation. Under serum-free, albumin-free culture conditions, the concentration of transferrin is usually 5.
It is used at 71 mg or more, on average 10 mg/'f. As is clear from FIG. 1, by adding a chelating agent, if there is only about one-tenth of the amount of transferrin as in the permanent state, cell proliferation can be improved by supplementing with trivalent iron. This is because the iron-transferrin conjugate (holo form) is taken into the cell via receptors on the cell surface, iron enters the cell, transferrin becomes the apo form, is expelled from the cell, and is returned to the trivalent form in the culture medium. This is because the chelating agent facilitates the so-called transferrin cycle, which binds to iron ions and is utilized for cell proliferation and metabolism. In other words, iron complexes created by chelating agents support cell proliferation even in the absence of transferrin, and in the presence of transferrin, promote the rotation of the transferrin cycle and significantly improve iron utilization efficiency. .

Test Example 3 Mouse hybridoma C2a was added to one volume of the culture medium of Example 3 described later.
- 5XlO' of E1 cells were seeded and cultured for 3 days at 37°C with 5% carbon dioxide gas in a 4-cell container.Then, the number of cells was returned to 5X10' and the culture was repeated in fresh medium of Example 3 for subculture. Ta. The results are shown in FIG.

Cells can be subcultured by addition of iron complexes in the absence of transferrin. This almost completely overturns the necessity of transferrin (conventional knowledge).

Test Example 4 Ferric chloride (hexahydrate) instead of transferrin 80
mg/j and the chelating agent shown in Table 2 below at the concentration shown in Table 2, and mouse hybridoma C2a-E I
The test was carried out and determined according to the procedure of Test Example 1 using cells. The results are shown in Table-2.

Chelating agents with a chelate stability constant of 15.87 or higher with trivalent iron ions have too strong a binding force with trivalent iron ions, making it impossible for cells to utilize iron ions stabilized as a complex. , cells do not proliferate. From the above results, it can be seen that the chelate stability constant of the trivalent iron ion of the chelating agent is preferably 15 or less.

Test Example 5 NLFK cells (feline The ability to support the growth of kidney cells) was determined according to the procedure of Test Example 1. However, since these cells are substrate-adherent cells, cell measurements were performed by peeling the cells from the vessel wall by trypsin treatment. The results are shown in Table-3.

Table 3 Average value and standard deviation of 4 main culture wells (number of XIO' cells/culture hole) The same effect of the chelating agent as on lymphoid cells (floating cells) was observed for adherent cells. That is, the effect of such a chelating agent suggests that its use can be expanded to general cultured cells.

Test Example 6 Mouse hybridoma cells R2- were added to 111 of the medium of Example 3 and the medium to which transferrin was added as a control.
5 x 104 Pt-E4-OB cells (producing anti-canine parvovirus antibodies derived from P3U-1 myeloma) are seeded and cultured in an incubator at 37°C with 5% carbon dioxide gas for 3 days. Thereafter, the antibody titer of the culture supernatant was semi-quantitated using enzyme immunoassay. The results are shown in FIG.

Antibody production equivalent to transferrin-added culture was observed in transferrin-free culture using a chelating agent. This indicates that when monoclonal antibodies are used for therapy in the future, highly pure antibodies can be obtained by reducing transferrin contamination during purification.

Test Example 7 105 NLFK cells and 1 unit of 10'-8 TCID cut/x1 unit of canine parvovirus were seeded together in the medium of Example 3 described later and Eagle's MEM medium supplemented with 10% tallow serum as a control, and incubated at 37°C for 8 days. The cells were cultured in a 5% carbon dioxide gas storage container, and the virus Ω of the culture supernatant was measured. The results are shown in FIG.

Virus yields comparable to or higher than conventional serum culture were observed. The application of iron complexes to kidney cells for vaccine production allows for the simplification of virus purification and is also useful in the industrial field of vaccine production.

Test Example 8 Each medium of Example 4.5 and the medium from which iron and chelating agent were removed from these formulations, or Eagle MEN supplemented with 10% bovine serum as a control, were each used at 3 and 1.5 yf in a 5 cm diameter culture dish. and add 2 normal human fibroblasts to this.
XIO' eggs were sown in a 5% carbon dioxide gas decompressor at 37°C for 5 days 7
% oxygen gas and 88% nitrogen gas. after that,
Cells were peeled off from the culture dish wall using trypsin, and the number of proliferating cells was counted. The results are shown in FIG.

Conventionally, in normal cells of this type, in the absence of transferrin, it sticks to the cell or vessel wall, and moreover, immediately after adding divalent iron (ferrous sulfate), which tilts the redox potential of the culture environment toward the reducing side. It is known that it only proliferates.

However, as shown here, just by adding trivalent iron as a complex to the culture solution in advance, they can proliferate, which was a new discovery that was previously unknown. The culture method based on this new concept of normal cells in an albumin-free, transferrin-free medium is particularly useful for research on the regulation of intracellular metabolic functions, as well as for anti-cancer and elucidation of the mechanisms of aging. It provides:

[Example] Hereinafter, the present invention will be explained in more detail with reference to Examples.
The present invention is not limited to these examples.

Example 1 Dissolve the following ingredients in twice-distilled water to prepare Solution A.

Ingredients Quantity L-Arginine 193.846mg
/ IL-arginine hydrochloride 152.248L
-Asparagine (monohydrate) 85.052L-Aspartic acid 19.384L-Cystine dihydrochloride 63.00OL-Cystine hydrochloride (monohydrate) 30.484L-Glutamic acid
19.384L-Glutamine 890
．． 770 Glutathione 0.970
Glycine 19.692L-Tyrosine 54.26OL-Histidine
14.5311L-Histidine Hydrochloride (1
water salt) 40.748L-hydroxyproline
19.384L-Inleucine 98.
912L-Leucine 98.912L
-Lysine Hydrochloride 109.594L-Methionine 29.092L-Phenylalanine 45.588L-Proline
29.384L-Serine 59
．． 078L-Threonine 95.954
L-tryptophan 14.548 L-valine 94.014 biotin
0.218 Calcium pantothenate
1.212 Choline hydrochloride 52
．． 908 Choline bitartrate 1.746 Folic acid 1.9401-inositol 35.864 Nicotinic acid amide
1.940 para-aminobenzoic acid
0.970 Pyridoxine hydrochloride 0.9
70 Pyridoxal Hydrochloride 0.970 Riboflavin 0.290 Thiamine Hydrochloride
1.940 dianocobalamin
0.00734 Monoethanolamine
40.000 Beef Insulin 20
．． 000 Putrescine dihydrochloride 0.025 Sodium pyruvate 220.000 Thymidine 0.025 Hyboxanthin
0.050 Sodium selenite
0.0034 glucose 3,
908.674 Sodium chloride 12,41
2.778 Potassium chloride 775.7
78 Calcium chloride 194.042 Calcium nitrate 67.362 Magnesium sulfate 138.014 Sodium hydrogen phosphate 77BJ54 Sodium dihydrogen phosphate
111.574 Sodium hydrogen carbonate 2,80
0.000 Kanamycin Sulfate 60.00
0 kanamycin 58.212 phenol red 10.888 then 4.436
g of ethylenediamine nibrobione hydrochloride to 8001
1 in double-distilled water and diluted with sodium hydroxide.
Adjust H to 7.2 to 7.4 (solution B). 0.2 to 20 g of ferric chloride (this amount may be adjusted as appropriate depending on the cells to be cultured) 1! 0. Dissolve in hydrochloric acid with O1 standard (liquid C). Mix 99 volumes of liquid B and 1 volume of liquid C to prepare liquid D. Mix 1 volume of solution A and 1 volume of solution D to obtain the desired serum-free medium.

Example 2 Hydroxyethyliminodiacetic acid 2.283 instead of ethylenediamine nibropionic acid hydrochloride in Example 1
A serum-free synthetic medium was prepared according to Example 1.

Example 3 A serum-free synthetic medium was prepared according to Example 1 by using 2.1.28 g of iminodiacetic acid in place of ethylenediamine nibrobione hydrochloride in Example 1 to prepare h solution.

Example 4 Dissolve the following ingredients in twice-distilled water to prepare Solution E.

Ingredients Amount Cmg/1) Eagle MEM 18.800 Amino acids L-Aspartic acid 26.6 L-Glutamine 58.4 Glycine
15.0L-glutamic acid 0.3L-proline 7.0L-serine
21.0 Vitamins, Hormones Folic Acid 0.001 Triiodothyronine 0.0004 Mouse Epidermal Growth Factor 0.02 Insulin 2.
0 Vitamin BI2 0.04 Biotin 0.04 Other organic acid salts Putrescine hydrochloride 0.04 Sodium pyruvate 220.0 Choline chloride
82. O Thymidine 0.14 Hypoxanthine 0.48 Trace element steel sulfate (pentahydrate) 0.00001 Manganese sulfate (7 hydrate) 0.0000048 Ammonium molybdate (monohydrate) 0.0024 Nickel chloride (6 hydrate) ) 0.000024
Ammonium metavanadate O,0OQ10B Selenite 0.00078 Buffer Sodium hydrogen carbonate 2,800 Sodium hydroxide 300 Next, add 0.1Bg of ferrous sulfate or 0.2g of ferric hydrochloride to 0.01 of 11 Dissolves in a specified amount of hydrochloric acid (G
liquid). Mix 99 volumes of solution B and 1 volume of solution F in Example 1 (solution H), and add 171,000 volumes of solution G!
Get the liquid. Mix 1 volume of Solution E and 1 volume of Solution 1 to prepare the desired serum-free synthetic medium.

Example 5 Solution B was prepared by using 2.283 g of iminodiacetic acid instead of ethylenediamine nibropionic acid hydrochloride in Solution B of Example 1, and a serum-free synthetic medium was prepared according to Example 4.

The serum-free synthetic medium prepared above is temporarily sterilized using a low protein adsorption membrane filter (pore size 0.22111, available as GV membrane from Millipore, USA), and stored in a cool, dark place while maintaining sterility. .

[Effects of the Invention] As explained above, according to the method of supplying iron to cells of the present invention, iron ions, which are essential for the proliferation of cultured cells and play an important role, are made into a stable complex. Iron ions can be taken into cells and made available for use without going through protein components such as albumin fractions and transferrin. Therefore, cells can proliferate and can be subcultured.

Furthermore, according to the serum-free synthetic medium of the present invention, since the iron compound is made into a stable complex using a chelating agent, iron is taken into the cells and utilized without becoming insolubilized, and the cells proliferate.
Subculture becomes possible. Therefore, there is essentially no need to add serum, albumin fraction, or expensive transferrin, which were conventionally required for compatibility testing, and a large quantity of medium with stable quality can be supplied at low cost. Furthermore, even when adding transferrin, the amount may be about one-tenth of the conventional amount.

Furthermore, since it is possible to culture cells using a completely serum-free synthetic medium that does not contain any serum proteins such as transferrin, it is possible to save time and effort when separating, purifying, and using cell products, and to develop a culture method based on new ideas. As a result, we can expect the development of new research methodologies.

[Brief explanation of drawings]

Figure 1 is a diagram comparing the effects of chelating agents, ferric chloride (hexahydrate), apotransferrin, and holotransferrin on the ability to support cell proliferation. Figure 2 is a diagram showing the state of cell proliferation in subculture according to the present invention. Figure 3 is a diagram showing the relationship between the amount of culture products of cells cultured according to the present invention and the amount of iron added, and Figure 4 is a comparison of the amount of virus in cultured Tsuchiura of canine balbovirus cultured according to the present invention. Figure 5 is a comparison diagram of the number of cultured normal human fibroblasts according to the present invention. Figure 2 Figure 3 Figure 5

Claims (1)

[Scope of Claims] 1) A method for supplying iron to cells, which comprises stabilizing iron with a chelating agent and supplying the stabilized iron to cells. 2) The method for supplying iron to cells according to claim 1), wherein the iron is trivalent and/or divalent iron ions. 3) The chelating agent is neutral to weakly alkaline and at least 2
The method for supplying iron to cells according to claim 1, wherein the chelating agent forms a more stable complex with trivalent iron ions than with valent iron ions. 4) The method for supplying iron to cells according to claim 1, wherein the chelating agent has a chelate stability constant of 15 or less with trivalent iron ions. 5) The method for supplying iron to cells according to claim 1), wherein the chelating agent is any one of ethylenediamine dipropioniohydrochloride, hydroxyethyliminodiacetic acid, and iminodiacetic acid. 6) The method for supplying iron to cells according to claim 1), wherein the concentration of the chelating agent is 0.1 to 15 mM. 7) The method for supplying iron to cells according to claim 1), wherein the cells are either animal cells or plant cells. 8) The method for supplying iron to cells according to claim 1, which is a method for supplying iron to cells in a synthetic medium. 9) The method for supplying iron to cells according to claim 8), wherein the synthetic medium is any one of a serum-free synthetic medium, a serum-free synthetic medium not added with transferrin, or a serum-free synthetic medium added with a small amount of transferrin. 10) A cell characterized by containing trivalent and/or divalent iron ions and a chelating agent that is neutral to weakly alkaline and forms a more stable complex with at least trivalent iron ions than with divalent iron ions. Serum-free synthetic medium for use. 11) Claim 10, wherein the chelating agent has a chelate stability constant of 15 or less with trivalent iron ions.
) The serum-free synthetic medium for cells described in section 2. 12) The serum-free synthetic medium for cells according to claim 10, wherein the chelating agent is any one of ethylenediamine dipropioniohydrochloride, hydroxyethyliminodiacetic acid, and iminodiacetic acid. 13) The serum-free synthetic medium for cells according to claim 10), wherein the concentration of iron ions is 0.05 to 10 mM. 14) The serum-free synthetic medium for cells according to claim 10), wherein the concentration of the chelating agent is 0.1 to 15 mM. 15) Basal medium is Eagle MEM, RPMI1640,
The serum-free synthetic medium for cells according to claim 10, which is Dulbecco's modified Eagle's medium, Ham's F12, or a mixed medium of two or more thereof.