JP2592144B2 - Nutrient for animal cell culture and method for producing the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION <Industrial Application Field> The present invention relates to a sterile nutrient for animal cell culture containing egg yolk low-density lipoprotein and a method for producing the same.

<Conventional technology> Conventionally, when culturing animal cells using a medium, for example, MEM medium, Iskov medium, RPMI1640 medium, Ham's F-12 medium, Tarbecco's modified MEM medium, Ham's F-12 medium A basal medium such as a 1: 1 mixture with a Tarbecco's modified MEM medium to which about 5 to 20% of fetal bovine serum (FBS) has been added to facilitate the growth of cells has been used. As described above, serum of bovine fetus and the like conventionally used as essential for cell growth is expensive. Therefore, a nutrient for animal cells capable of greatly reducing the use ratio of this serum is required. ing.

By the way, it is known that the yolk low-density lipoprotein has an excellent cell growth effect, and can be used in combination with a growth factor such as insulin or transferrin to prepare a serum-free medium for some cells including hybridomas. I have.

However, eggs produced from parent birds infected with viruses or mycoplasmas as raw eggs are often said to be contaminated with these microorganisms. This is the so-called vertical transmission. Moreover, chicken viruses and mycoplasmas are said to be widespread worldwide. Therefore, generally, when yolk low-density lipoprotein is prepared using commercially available eggs, there is a great risk that the final product is contaminated with virus or mycoplasma. In addition, most of these components pass through in sterile filtration using a microfilter, which is usually performed, so that when the raw eggs are contaminated, they cannot be removed by sterilization alone. Further, there is a problem that when heat-sterilizing egg yolk low-density lipoprotein at a high temperature, turbidity occurs, and when used as a nutrient for animal cells, the medium becomes turbid.

<Problems to be Solved by the Invention> By the way, viruses and mycoplasmas inhibit the growth of animal cells and change the properties of animal cells. Should of course not contain viruses or mycoplasmas.

Therefore, conventionally, when yolk low-density lipoprotein is used as a nutrient for animal cell culture, parent birds are bred aseptically so as not to be infected with virus or mycoplasma, and egg yolk low-density lipoprotein is prepared from eggs of the parent bird as a raw material. It must be extracted and used as a nutrient for culturing animal cells, which requires special equipment, and has the problem of becoming an expensive nutrient.

In view of such circumstances, an object of the present invention is to provide a nutrient for culturing animal cells which contains an egg yolk low-density lipoprotein, is sterile, and can be easily and inexpensively produced, and a method for producing the same.

<Means for Solving the Problems> The nutrient for culturing animal cells according to the present invention for achieving the above object is a sterile nutrient for culturing animal cells, comprising yolk low-density lipoprotein and 1.9% by weight or more of sodium chloride. The method is characterized in that the yolk low-density lipoprotein is dissolved in saline to obtain a solution containing 1.9% by weight or more of sodium chloride.
It is characterized by performing a heat sterilization treatment at 70 ° C. and a microfilter overtreatment.

Here, the yolk low-density lipoprotein is a fraction of the yolk protein that floats when a yolk solution having a specific gravity of about 1.063 with sodium chloride and water is centrifuged. The above-mentioned yolk low-density lipoprotein referred to in the present invention is typically obtained by removing the granule fraction that precipitates when centrifuged by adding an equal volume or twice the volume of a 0.15 mol aqueous sodium chloride solution to the yolk, Sodium chloride is added to the supernatant to adjust the specific gravity to about 1.063, and it is obtained as a floating fraction (abbreviation: LDL) when centrifuged (usually at 30,000 rpm for 16 to 24 hours). Is dissolved in an aqueous solution of 10% by weight of sodium chloride (hereinafter, all percentages are simply expressed as%), and then the specific gravity of the solution is adjusted to about 1.06 by adding sodium chloride.
Adjust to 3 and fraction that floats when centrifuged (abbreviation: LD
LG). These yolk low-density lipoproteins collectively referred to as LDL and LDLG are hereinafter abbreviated as egg yolk LDL.

In order to obtain a large amount of this yolk LDL by a single centrifugation, the following treatment may be performed. That is, 2 times the amount of 0.
A 15 molar sodium chloride solution was added and stirred gently.
Dialyze for 72 hours. After dialysis, the precipitate was removed by centrifugation at 10,000 rpm for about 1 hour, and an equal amount of distilled water was added to the supernatant.
Adjust the pH to 6.0 with a 1 / 50N hydrochloric acid solution. Then yolk L
As DL comes up naturally, leave it overnight at 4-6 ° C and collect the things that come up. If you are in a hurry, after adjusting the pH as described above, immediately centrifuge at 5,000 to 10,000 rpm for about 30 minutes to collect the floating substances.

Egg yolk LDL has a lipid content of usually 84
~ 89%, with a protein content of about 11-16%.

The raw material egg of the yolk LDL obtained in this manner may be a commercially available egg of birds such as chicken, quail, and duck. When collecting egg yolk LDL from a raw egg, it is desirable to use a sterilized instrument in a clean bench so as to prevent contamination by various bacteria.

The present invention provides a sterile nutrient for animal cell culture using such egg yolk LDL. Here, aseptic refers to a state in which mycoplasma has been killed, the virus has been inactivated, and general live bacteria (bacteria) have been killed or removed.

In the present invention, by coexisting 1.9% or more of salt with egg yolk LDL, the temperature at which egg yolk LDL starts heat denaturation during heat sterilization, that is, the temperature at which turbidity starts to occur, is increased, thereby producing turbidity. Without heating, mycoplasmas and viruses can be completely sterilized by heating.

In the present invention, the salt content of 1.9% or more is about 6%.
In order to obtain the effect that mycoplasma and viruses can be completely sterilized without causing turbidity under heating conditions of 0 to 70 ° C., as shown in the test described below when the salt content is less than 1.9%, for example, Heat sterilization at 60 ° C. for 3 hours and at 65 ° C. for 1 hour causes turbidity, and the above effects cannot be obtained. On the other hand, the same effect can be obtained by using sodium chloride until it reaches the saturation concentration. Also, since such nutrients are only added in very small amounts to the base medium,
As described below, a high salt concentration has no effect on cell culture.

On the other hand, the content of egg yolk LDL in the nutrient for animal cell culture of the present invention may be adjusted to a practical concentration of 1 to 10% as a nutrient.

Next, a preferred method for producing the nutrient for animal cell culture of the present invention will be described.

In order to obtain the nutritional supplement for animal cell culture of the present invention, first, egg yolk LDL is dissolved in, for example, a saline solution having a concentration of 2% or more (or a saturated saline solution) to a concentration of 1 to 10%. And
By heating this solution at a temperature of 60 to 70 ° C.,
Completely kills mycoplasmas and viruses without turbidity. Next, the mixture is passed through a microfilter to remove general viable bacteria that do not die under the above heating conditions, thereby obtaining a completely aseptic nutrient for animal cell culture.

Here, the heat sterilization conditions vary depending on the salt concentration in the nutrient, etc.
For 1 to 6 hours, and when the salt concentration is 6 to 11%,
Any condition such as a condition for 12 hours that can sterilize or inactivate mycoplasma or virus without causing turbidity may be used. If the temperature is lower than 60 ° C., complete sterilization or inactivation of mycoplasma or virus cannot be performed, and if the temperature is higher than 70 ° C., turbidity and coagulation of the nutrient are likely to occur, which are both undesirable.

The nutrient for animal cell culture of the present invention thus obtained is easily oxidized during storage because its pH is 4 to 6, and when stored for a long period of time, oxidation progresses and the growth effect of animal cells is reduced. There is a tendency. Therefore, in order to prevent oxidation during storage and maintain the growth effect, an alkaline agent such as caustic soda and caustic potash is added to the obtained nutrient for animal cell culture, and the pH of the nutrient is 7 or more, preferably 7 to It is good to be 11. If the pH of the nutrient exceeds 11, egg yolk LDL tends to coagulate, so it is desirable that the pH be 11 or less.

Egg yolk LDL was dissolved in saline to reduce the salt concentration to 1.9%.
After passing through a microfilter, the solution
Similar nutrients can be obtained by heating to ~ 70 ° C.

<Test Example> Test Example 1 A 1.5-fold volume of a 0.15 mol aqueous sodium chloride solution was added to egg yolk, and the precipitate was removed by centrifugation. The specific gravity obtained by adding sodium chloride to the supernatant thus obtained was adjusted to about 1.063,
Egg yolk LDL was obtained by centrifugation at 000 rpm for 16-24 hours.

Next, 5% of concentration 2.0%, 2.7%, 5.4%, 10.8%, 16.8%
Egg yolk LDL was dissolved 5% each in each kind of saline, and used as a nutrient for animal cell culture. These are designated as specimens 1 to 5,
The salt concentration in the nutrients of each test product 1 to 5 is 1.9%, 2.57%,
5.13%, 10.3% and 15.4%.

For comparison, 5% yolk LDL was added to a 1.8% saline solution.
It was dissolved to obtain a comparative product (the salt concentration in the nutrient was 1.71%).

Each of these test products 1 to 5 and the comparative product was separately heated under the conditions of temperature and time shown in Table 1, and the state thereafter was observed. The results shown in Table 1 were obtained.

As shown in Table 1, the comparison product with a salt concentration of 1.71%
Although turbidity was observed under the heating condition of 3 hours at 65 ° C. and 1 hour or more at 65 ° C., the conditions for disinfecting viruses and mycoplasmas can be selected in the range of 60 to 70 for the test products 1 to 3 according to the present invention. At a heating temperature of 75 ° C., it was recognized that even if the salt concentration was increased, turbidity occurred and proper sterilization could not be performed.

Test Example 2 Serum-free using test samples that did not show turbidity under the heating conditions of 65 ° C and 70 ° C in Test Example 1 (including those in which slight turbidity did not affect the observation of the culture medium) The medium was adjusted.

The serum-free medium used was a mixture of equal amounts of Ham's F-12 medium and Dulbecco's Iscove's modified medium as the basic medium, and 200 µg / ml of each test product of the above nutrient, insulin 6
μg / ml, transferrin 6 μg / ml, bovine serum albumin
It was adjusted by adding 1 mg / ml and 6 mg / ml selenium.

As a control, a medium supplemented with 5% fetal bovine serum (FBS) was prepared.

Mouse myeloma was inoculated in each of the above media and grown at an initial cell number of 3 × 10 ⁴ / ml, and the number of cells was counted. The results in Table 2 were obtained. The values in the table are 5% FBS
This is a relative value when the number of cells in the supplemented medium is 100.

From the results shown in Table 2, each test product according to the present invention showed a cell growth effect equivalent to that of expensive fetal bovine serum (FBS), and the nutrient of the present invention was contaminated with virus and mycoplasma due to its favorable growth condition. It is inferred that it has not been done. In addition, since only a small amount of each test article was added to the basal medium, it was confirmed that a high salt concentration did not affect cell culture.

Test Example 3 50 ml of a test product (nutrient for animal cell culture) in which no turbidity was observed under the heating condition of 75 ° C. in Test Example 1 was divided into eight equal parts, each having a pH of 3.0, 4.3, 5.2, 5.9, 6.8, 7.8. , 8.3 and 9.3 were prepared. In addition, a diluted hydrochloric acid aqueous solution or a diluted caustic soda aqueous solution was used for pH adjustment of each sample.

(1) Each sample was filled and sealed in a test tube with a screw cap (10 ml volume) in a volume of 5 ml for each sample, and then stored in a thermostat at 25 ° C, and the degree of oxidation of the sample after 10 days and 30 days was measured. It was measured. The degree of oxidation is compared by the peroxide value (POV) of each sample, and the value is shown as the absorbance at a wavelength of 500 nm measured by a spectrophotometer. That is, the higher the absorbance, the more the oxidation progresses. The result is shown in FIG.

As shown in FIG. 1, when the nutrient for animal cell culture according to the present invention was adjusted to pH 7 or more, it was recognized that oxidation hardly progressed during storage.

(2) A serum-free medium was prepared in the same manner as in Test Example 2 using each sample after storage for 30 days in (1), and NS-1 cells were inoculated in each medium (initial cell count). 3 × 10
⁴ / ml), and the cells were counted after 7 days and 14 days to examine the growth of cultivation. The result is shown in FIG. The growth rate 100 in the figure indicates the growth rate when 5% FBS (fetal calf serum) was used.

As shown in FIG. 2, the state of cell proliferation in each sample is almost inversely proportional to the degree of oxidation of each sample. That is, the sample with a higher degree of oxidation had a lower cell growth effect, whereas the sample with a lower degree of oxidation had a good cell growth state.

As described above, it was confirmed that when the pH of the nutrient was adjusted to 7 or more, oxidation hardly proceeded during storage and the cell growth effect did not decrease.

<Effects of the Invention> As described above, according to the present invention, a simple and inexpensive, sterile, non-turbid yolk LD can be obtained from commercially available eggs with shells.
It can be a nutrient for animal cell culture containing L. Moreover, this nutrient has the same cell growth effect as expensive bovine fetal serum and is sterile, so that animal cells can be cultured without changing the properties of the cells.

Further, when the nutritional supplement of the present invention has a pH of 7 or more, it is possible to provide a product which is hardly oxidized during storage and whose cell growth effect does not decrease even after long-term storage.

[Brief description of the drawings]

FIG. 1 is a graph showing the PH of the sample of Test Example 3 and the degree of oxidation during storage, and FIG. 2 is a graph showing the growth state of the cells of each sample of Test Example 3.

Claims (3)

(57) [Claims] 1. A sterile nutrient for animal cells, which is a solution containing low-density lipoprotein of yolk and 1.9% by weight or more of sodium chloride. 2. The nutritional supplement for animal cells according to claim 1, wherein the pH of the solution is 7 or more. 3. A solution containing yolk low-density lipoprotein in saline solution to obtain a solution containing 1.9% by weight or more of sodium chloride, and then subjecting the solution to heat sterilization at 60 to 70 ° C. and passing through a microfilter. A method for producing a nutrient for animal cell culture, characterized in that: