JPH08308561A - Serum-free medium for animal cell culture - Google Patents

Abstract

PURPOSE: To obtain the subject medium by adding a water-soluble selenium compound to a medium, capable of excellently culturing a cell without adding a serum and without losing functionality of the cell, having slight variability between products due to inter-lots. CONSTITUTION: A medium is blended with 10<-6> to 10<-7> M of a selenium compound such as a selenate or a selenite (e.g. sodium selenite, etc.), 1-2vol.% of a cell membrane stabilizer such as dimethyl sulfoxide(DMSO), etc., 10<-6> to 10<-7> M of glucocorticoid such as dexamethasone, etc., as a cell function improver, 10<-6> to 10<-7> M of insulin, 10-50ng/ml of one or more of epidermal growth factor(EGF), platelet-derived growth factor(PDGF), fibroblast growth factor(FGF), hepatocellular growth factor(HGF), etc., as a cell growth factor, 20-50μg/ml of proline, etc., to give the objective serum-free medium for animal cell culture, capable of excellently culturing a cell without adding a serum and without losing functionality of the cell, having slight variability between products due to inter-lots.

Description

Detailed Description of the Invention

[0001]

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

[0002]

2. Description of the Related Art In recent years, in the field of biochemistry, experiments using animal cells have been carried out to elucidate biological functions, and methods for culturing animal cells have become increasingly important.

In order to maintain the cell function of cultured animal cells for a long period of time, it is necessary to add serum to the medium. Therefore, it is necessary to add serum such as fetal bovine to the medium at a concentration of about 10%. Must be used.

On the other hand, the method of adding serum to the medium has the following problems. 1 In the serum to be added, there is a possibility that a large number of unknown impurities, whose main function is protein and whose action function is unknown, are mixed. The composition of various unknown impurities is different and the performance of the medium is not constant, which causes a problem in reproducibility. 2 A potential source of viruses and mycoplasma contamination.

For this reason, a method of reducing the serum concentration in the medium can be considered. However, if the serum is too small, the functional decline or death of the cells will be caused, and therefore there is a limit to the reduction of the serum concentration in the medium.

[0006]

Therefore, in order to solve the above problems, the cells are composed of only known components that can be satisfactorily cultured without loss of functionality without the addition of serum. Development of medium is strongly desired.

[0007]

Means for Solving the Problems Accordingly, the present inventors have:
In order to solve the above problems, earnest studies have been made, and an animal cell culture serum-free medium capable of maintaining cell functions without serum for a long period of time can be obtained by adding and mixing only known substances to a synthetic basic medium. Heading The present invention has been completed. That is, the present invention provides a serum-free medium for animal cell culture, which has the same effect as the above-mentioned serum-containing medium, by containing a selenium-containing compound in the medium and further mixing a known substance such as DMSO in the medium.

Therefore, the present invention, in particular, is combined with a collagen gel culture method using, for example, type I collagen from which telopeptide has been removed as a culture substrate, so that both the medium and the culture substrate contain unknown substances in the culture system. Therefore, the present invention provides a serum-free medium for animal cell culture, which makes it possible to simply set up an experimental system for cell function, drug metabolism, and toxicity test by drugs. Furthermore, the present invention does not need to consider the influence of dozens of types of proteins and hormones in the culture substrate or serum components at all, and animal cell cultures that enable long-term tests based on long-term cultures. A serum-free medium for use is provided.

More specifically, the present invention relates to a serum-free medium for animal cell culture, characterized in that the medium contains a selenium compound.

The present invention also relates to a serum-free medium for culturing animal cells, which contains a water-soluble selenium compound.

Further, the present invention relates to a serum-free medium for animal cell culture, which is selenite or selenate as a water-soluble selenium compound.

The present invention also relates to a serum-free medium for culturing animal cells, which contains the selenium compound in an optimum concentration.

Further, in the present invention, the selenium compound is at least (1/100) times or more and 100 times or less the optimum concentration,
The present invention relates to a serum-free medium for culturing animal cells, which is contained in a ratio of (1/10) times or more and 10 times or less.

The present invention also relates to a serum-free medium for animal cell culture, characterized in that the medium further contains a cell membrane stabilizer.

Further, the present invention relates to a serum-free medium for animal cell culture, wherein the cell membrane stabilizer is dimethyl sulfoxide.

The present invention also relates to a serum-free medium for animal cell culture, characterized in that the medium further contains a cell growth factor.

The present invention further provides, as a cell growth factor, E
It relates to a serum-free medium for animal cell culture, which is one selected from the group consisting of GF, PDGF, FGF and HGF.

Furthermore, in the present invention, the cell growth factor is EGF
The present invention relates to a serum-free medium for animal cell culture, characterized in that

The present invention also relates to a serum-free medium for animal cell culture, characterized in that the medium further contains a cell function promoter.

The present invention further relates to a serum-free medium for animal cell culture, wherein the cell function promoter is glucocorticoid or insulin.

Further, the present invention relates to a serum-free medium for animal cell culture, characterized in that it is dexamethasone or dexamethasone and insulin as glucocorticoids.

In the present invention, sodium selenite is added to the medium at 10 ^-6 to 10 ^-7 M and DMSO is added at 1 to 2% by volume.
, Dexamethasone 10 ^-6 to 10 ^-7 M, insulin 10 ^-6 to 10 ^-7 M, and EGF 10 to 50 ng / ml.
And a serum-free medium for animal cell culture, which contains at least 20 to 50 μg / ml of proline.

The present invention will be described in more detail below. (Medium) The medium that can be used in the present invention is not particularly limited, and a medium that can be generally used as a medium for animal cells can be preferably used. For example, in hepatocytes, L-15 medium, William's E medium, Waymouth's MB
752/1 medium and the like can be used particularly preferably.

Further, the medium can also usually contain a carbon source which can be assimilated by a culture medium such as animal cells, a nitrogen source which can be digested, and an inorganic salt. Moreover, a trace amount effective substance such as a trace nutrient promoting substance or a precursor may be blended as necessary. Here, glucose, mannitol, glycerol, fructose and the like can be used as the carbon source, and organic nitrogen compounds such as casein and BSA, and inorganic nitrogen compounds such as sodium nitrate and ammonium sulfate can be used as the nitrogen source. .

(Selenium Compound as Antioxidant) The type of selenium compound that can be used as an antioxidant in the present invention is not particularly limited. Selenium is found in glutathione peroxidase (glutathi peroxidase) in hepatocytes and other tissue cells.
one-peroxidase (GSH-Px), it is known to be contained in the form of selenocysteine and replace S (sulfur) of cysteine in vivo, and further, the substituted glutathione peroxidase (selenium) is produced in cells. It is known to stabilize biological membranes by degrading lipid peroxides (Nutritional Chemistry, Hiroshi Naito et al., Asakura Publishing).

Therefore, in the present invention, various selenium compounds capable of substituting and exerting the above-mentioned action of stabilizing the biological membrane,
A water-soluble selenium compound is particularly preferable, and for example, selenium oxide or selenium oxide ion can be used. Further, for example, in the present invention, selenious acid or a salt thereof (eg, alkali metal salt, alkaline earth metal salt), selenic acid or salt thereof (eg, alkali metal salt, alkaline earth metal salt), etc. are preferable. It can be used.
In the present invention, particularly, selenite, for example, sodium selenite can be more preferably used for culturing hepatocytes.

(Optimal Concentration of Selenium Compound) In the present invention, the concentration used of the selenium compound is
And can be optimized depending on the type of cells to be cultured,
There is no particular limitation on the optimization method. For example, an increase in the number of cultured cells due to a change in the concentration of the selenium compound (the increase in the number of cultured cells can be counted by, for example, observation under a hemocytometer and a microscope, colony formation method, etc.), or production of cultured cells The optimum concentration (for example, maximum peak concentration) can be suitably estimated by measuring the increase in the substance.

For example, in the case of hepatocytes, DMSO, EGF and other components are added to an appropriate medium, and sodium selenite is added at a final concentration (concentration when used as a medium) of 10 ^−3. Hepatocytes were cultured by adjusting a plurality of mediums added so as to have a concentration of ^{-10 -12} M. For example, albumin produced by hepatocytes was analyzed by an enzyme-linked immunosorbent assay (ELISA method).
It is possible to estimate the concentration of sodium selenite when the production amount of the albumin shows the maximum value as the optimum concentration. Furthermore, in the present invention, the optimum concentration estimated by the above method is about 1
It can be preferably used even in a concentration range of / 100 to 100 times. Further, in the present invention, it can be preferably used in a concentration range of about 1/10 to 10 times the optimum concentration estimated by the above method.

For example, in the case of hepatocytes, the optimum concentration is estimated to be 10 ⁻⁶ to 10 ⁻⁷ M, and therefore 10 ⁻⁵ to 10 ⁻⁸ M.
Can be suitably used at a concentration of 10 ^-4 to 10
It is shown that it can be used even at a concentration of ^-9 M.
If the concentration is higher than this (for example, the optimum concentration above 100)
More than twice) is not preferable because cytotoxicity occurs.
If the concentration is too low (for example,
If it is 100 times or less), the above-mentioned action is not sufficiently exhibited, which is not preferable.

In the present invention, the optimum concentration of the selenium compound means the concentration when used for cell culture, and does not have to be the concentration before adjustment of the medium or during storage. . Further, in the present invention, the optimum concentration does not depend on the mode of use of the medium. That is, it is not limited to the form of the aqueous solution, and it is possible to use it after diluting it or to use it by gelling it by another method, and the concentration at the time of use may be the above-mentioned optimum concentration.

Further, as a method for detecting the selenium compound in the medium, a general selenium compound analysis method can be used. For example, for the confirmation and quantification of the presence of selenium element, atomic absorption spectrometry, Analysis methods such as ion-coupled plasma mass spectrometry and ion column chromatography can be preferably used. In the analysis of ion species,
Various ion chromatographic methods and the like can be used.

(Cell Membrane Stabilizer) In the present invention, it is effective to add a cell membrane stabilizer together with the selenium compound as the antioxidant. In the present invention, the cell membrane stabilizing agent is not particularly limited, but dimethyl sulfoxide (DMSO) or a compound having a cell membrane stabilizing property equivalent to DMSO can be preferably used. The use of DMSO is particularly preferred in the present invention.

The cell membrane stabilizer is used for long-term culture.
It is considered to have the effect of stabilizing the cell membrane of cultured cells and protecting it from various external influences. The concentration of the stabilizer used can be optimized depending on the stabilizer used and the type of cells and is not particularly limited. Generally, a constant concentration is required to sufficiently exhibit the above-mentioned action. For example, in animal cells, the final concentration is about 0.1.
It is preferably added in an amount of 2 wt%. For example, in the case of hepatocytes, when DMSO is used, the final concentration is preferably about 0.1-2% by weight. That is, if the concentration is lower than this, sufficient cell membrane stabilizing effect is not exhibited, and if the concentration is too high, cytotoxicity or the like occurs, which is not preferable.

Further, as a method for detecting the stabilizer in the medium, a general method for detecting an organic substance can be used. For example, gas chromatograph, column chromatograph, infrared absorption spectrum, nuclear magnetic resonance absorption spectrum, etc. It can be preferably used.

(Cell Growth Factor) In the present invention, it is preferable to use a cell growth factor in combination with the above-mentioned antioxidant and cell membrane stabilizer. Use of the factor enables stable cell growth for a long period of time. In the present invention, what can be used as a cell growth factor is not particularly limited, but for example, epidermal growth factor (epidermal growth factor:
EGF), platelet-derived growth factor (platelet-derived gro
wth factor: PDGF, fibroblast growth factor (fibrob
lase growth factor (FGF), hepatocyte growth factor (hepa)
tocyte growth factor (HGF) and the like can be particularly preferably used. For example, in the case of hepatocytes, EGF,
HGF and the like can be preferably used.

The concentration of the factor to be used can be optimized depending on the factor to be used and the type of cell and is not particularly limited. Generally, it is sufficient to add a final concentration of about 10 to 100 ng / ml in animal cells. For example, in the case of hepatocytes, when EGF is used, the final concentration is preferably 10 to 50 ng / ml. That is, when the concentration is lower than the above concentration, sufficient cell growth action is not exhibited, and when the concentration is too high, the activity is saturated, the desired effect cannot be expected, and there is a problem such as decomposition due to an increase in protein components. Can occur.

Further, the method for detecting the factor in the medium is
Although not particularly limited, the immunoassay method can be preferably used since the concentration is very small and many low-concentration coexisting substances are present. For example, in the case of EGF, EGF can be obtained by a radioimmunoassay method (RIA) using an EGF antibody, an enzyme immunoassay method (EIA, ELISA), or the like.
Can be analyzed up to a concentration of about 100 pM.

(Cell Function Promoter) Further, in the present invention, it is possible to use a cell function promoter together with the cells to be cultured. The type and concentration of the promoter can be optimized and selected according to the type of cell. For example, in animal cells, glucocorticoids can generally be preferably used. Furthermore, among the glucocorticoids, cortisol, corticosterone, cortisone, dexamethasone, prednisolone and the like can be preferably used, and particularly in the case of hepatocytes, dexamethasone and the like as a liver function promoter can be preferably used. .

Further, in the present invention, a combination of insulin-like growth factor (IGF) and the like can be preferably used for promoting the function of cultured cells. For example, in hepatocytes, insulin has actions such as suppressing gluconeogenesis, promoting glycogen synthesis, and promoting protein synthesis.

(Mode of Use) In the present invention, the mode of use of the medium is not particularly limited. Aspects of an aqueous solution, modes of use after gelation, immobilization of the above-mentioned medium components, or dissolution of a semi-solid mixture in a necessary amount of an aqueous solution, or use after gelation are suitably possible.

[0041]

[Function] By adding a selenium compound as an antioxidant to the medium to decompose peroxides and the like produced in cells to enable stable long-term culture, and by further adding a cell membrane stabilizer to the medium, It protects the animal cell membrane, enables stable long-term culture, and if necessary, adds a cell growth factor or a cell function promoter to help the growth of animal cells and enhance cell function, thereby completely eliminating serum. A medium is obtained to allow long-term animal cell culture without the need for use.

[0042]

EXAMPLES The present invention will be specifically described below based on examples, but the present invention is not limited to the following examples as long as the gist thereof is not exceeded. (Example 1) Determination of optimum concentration of sodium selenite Preparation of cell culture vessel A pepsin-soluble collagen solution was added to a reconstitution buffer (0.05% sodium hydroxide, 4.77%) at a low temperature of 4 ° C.
HEPES, 2.2% sodium hydrogen carbonate) and a 10-fold concentrated culture solution were added at a ratio of 8: 1: 1 (volume ratio), mixed well to neutralize, and uniformly injected into the bottom of the culture vessel. Then, heat it in an incubator and gel it before use.

B. Preparation of serum-free medium In L-15 medium as a medium, insulin 10 ^-7 M, EG
F10 ng / ml, DMSO2%, dexamethasone 10
^-7 M and proline 30 μg / ml were added, and sodium selenite was further added at 10 ⁻³ to 10 ^{−12, respectively.}
After the addition of M was adjusted, it was sterilized by filtration using a 0.22 μm filter. Further, as a control medium, the medium without the addition of DMSO and EGF was used.

C. Culture of hepatocytes The following cell culture was performed using the cell culture container and the serum-free medium prepared by the above method. Hepatocytes from rats were subjected to collagenase perfusion method (protein, nucleic acid, enzyme, 1978, 2
3, 1259-1271), and 5 × 10 ⁵ cells / ml on the gel in the collagen gel culture vessel.
The seeds were seeded at a density of and cultured at 37 ° C. in a 5% carbon dioxide gas incubator.

The above-mentioned serum-free medium was used as the culture medium, the culture medium was added to the upper surface of the gel layer, and the medium was replaced every 2 to 3 days to continue the culture. The amount of secreted albumin synthesized by hepatocytes was measured by periodically exchanging the culture medium and measuring albumin in the culture medium on the 21st day of culture. The ELISA method was used for the measurement of albumin. The result is shown in Figure 1.
As shown in. It was confirmed that when the sodium selenite concentration was 10 ⁻⁶ to 10 ⁻⁸ M in a medium containing EGF and DMSO, the albumin production amount of the cultured hepatocytes reached a peak and the cell function was maintained. Therefore, it was found that the serum-free medium containing this optimum concentration of selenite can maintain the cell function for a long period of time.

(Example 2) Comparison with serum medium A. Preparation of cell culture vessel A pepsin-soluble collagen solution was added to a reconstitution buffer (0.05% sodium hydroxide, 4.77%) at a low temperature of 4 ° C.
HEPES, 2.2% sodium hydrogen carbonate) and a 10-fold concentrated culture solution were added at a ratio of 8: 1: 1 (volume ratio), mixed well to neutralize, and uniformly injected into the bottom of the culture vessel. Then, heat it in an incubator and gel it before use.

B. Preparation of serum-free medium In L-15 medium as a medium, insulin 10 ^-7 M, EG
F10 ng / ml, DMSO2%, dexamethasone 10
^-7 M and proline (30 μg / ml) were further added with sodium selenite (10 ^-6 M), and the mixture was sterilized by filtration using a 0.22 μm filter. In addition, a medium to which sodium selenite was not added was used as a serum-free medium for comparison, and a medium to which 10% fetal bovine serum was added instead of sodium selenite was used as a serum medium.

C. Culture of hepatocytes The following cell culture experiment was performed using the cell culture container and the serum-free medium prepared by the above method. Hepatocytes were separated from rats by the collagenase perfusion method, seeded on the gel in the collagen gel culture vessel at a density of 5 × 10 ⁵ cells / ml, and cultured at 37 ° C. in a 5% carbon dioxide gas incubator. The above-mentioned serum-free medium was used as the culture medium, the culture medium was added to the upper surface of the gel layer, and the medium was replaced every 2-3 days to continue the culture. The culture solution was exchanged and sampled periodically for 4 weeks, and albumin in the culture solution was measured (by the ELISA method) to measure the amount of albumin secreted by hepatocytes.

The results are shown in FIG. When cultivated in a medium containing sodium selenite, it was confirmed that long-term culturing of cells comparable to serum medium was able to maintain the function, while long-term culturing was not possible in sodium selenite-free medium. It was As a result, it was found that the serum-free medium containing selenite can maintain the cell function for a long period of time.

[0050]

INDUSTRIAL APPLICABILITY As described above, the present invention provides a medium for culturing animal cells, which has the same effects as the conventional serum medium and does not require serum at all. Further, the animal cell culture medium according to the present invention, the constituent material of the culture medium is composed only of known substances, does not cause variations in the characteristics between the culture medium due to the difference in lots seen in serum culture, animals with good reproducibility Cell culture becomes possible.

[Brief description of drawings]

FIG. 1 is a graph showing the albumin secretion amount of hepatocytes cultured in a medium containing various sodium selenite concentrations measured in Example 1.

FIG. 2 is a diagram showing changes in albumin secretion of cultured hepatocytes measured in Example 2.

Claims (16)

[Claims] 1. A serum-free medium for animal cell culture, characterized in that the medium contains a selenium compound. 2. The serum-free medium for animal cell culture according to claim 1, wherein the selenium compound is water-soluble. 3. The serum-free medium for animal cell culture according to claim 2, wherein the water-soluble selenium compound is selenite or selenate. 4. The serum-free medium for animal cell culture according to claim 1, wherein the medium contains the selenium compound in an optimum concentration. 5. The animal cell culture non-use cell according to claim 1, wherein the medium contains the selenium compound at least (1/100) times or more and 100 times or less of an optimum concentration. Serum medium. 6. The animal cell culture non-use cell according to claim 1, wherein the medium contains the selenium compound at least (1/10) times or more and 10 times or less the optimum concentration. Serum medium. 7. The serum-free medium for animal cell culture according to claim 1, wherein the medium further contains a cell membrane stabilizer. 8. The serum-free medium for animal cell culture according to claim 7, wherein the cell membrane stabilizing agent is dimethyl sulfoxide. 9. The serum-free medium for animal cell culture according to claim 1, wherein the medium further contains a cell growth factor. 10. The cell growth factor is epidermal growth factor (EGF), platelet-derived growth factor (PDGF),
Fibroblast growth factor (FG)
F), hepatocyte growth factor: H
The serum-free medium for animal cell culture according to claim 9, which is at least one selected from the group consisting of GF). 11. The serum-free medium for animal cell culture according to claim 10, wherein the cell growth factor is EGF. 12. The serum-free medium for animal cell culture according to claim 1, wherein the medium further contains a cell function promoter. 13. The cell function promoter is a glucocorticoid or insulin.
2. A serum-free medium for culturing animal cells according to 2. 14. The serum-free medium for animal cell culture according to claim 13, wherein the glucocorticoid is dexamethasone. 15. The cell function promoting agent is dexamethasone and insulin.
A serum-free medium for culturing animal cells according to item 1. 16. A medium containing 10 parts of sodium selenite.
^-6 to 10 ^-7 M, DMSO 1 to 2% by volume, dexamethasone 10 ^-6 to 10 ^-7 M, and insulin 10 ^-6 to 1
A serum-free medium for animal cell culture, which contains at least 0 ⁻⁷ M, EGF at 10 to 50 ng / ml, and proline at 20 to 50 μg / ml.