JP3081999B2 - Culture medium - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel medium for animal cells.

[0002]

2. Description of the Related Art To culture animal cells, 2 to 20% of animal serum is generally added to a basic synthetic medium consisting of various inorganic salts, saccharides, amino acids, vitamins, etc. to supplement nutrients.
Added serum-containing media has been used.

[0003]

However, fetal bovine serum, which is used as an essential ingredient for cell growth, is very expensive because it uses fetal bovine blood as a raw material. If an inexpensive alternative substance can be used, it is advantageous in making the medium for animal cells inexpensive. Therefore, there is a demand for a substance capable of greatly reducing the use ratio of fetal bovine serum in the medium.

Recently, low-density lipoprotein (LDL) of egg yolk has been added to ham F-12 medium or Iscove's modified Dulbecco's medium (IMDM medium) which is a basic medium, and if necessary, insulin, transferrin, selenium, etc. Serum-free media to which growth factors such as brain serum albumin (BAA) and linoleic acid and nutrient supplements have been added have come to be seen. However, at present, it is not always sufficient to use the above-mentioned nutrients, and the appearance of various nutrients for animal cells is desired.

In view of such circumstances, the present inventors have conducted intensive studies on substances which can significantly reduce the amount of serum used, such as fetal bovine serum, when used in a basal medium. They found a substance suitable for culturing animal cells, and invented a novel medium for animal cells.

[0006]

The gist of the present invention is to add an animal thymus extract to a medium.

In the present invention, the term "basal medium" refers to a medium composed of various inorganic salts, sugars, amino acids, vitamins, and the like generally required by cells. Specifically, Eagle MEM
Medium, 197 medium, Dulbecco's modified Eagle medium (DM
EM medium), Iskov medium, Iskov modified Dulbecco medium (IMDM medium), Ham F-10 medium, Ham F-12
Media, RPMI-1640 medium and the like. In addition, those containing a small amount of fetal bovine serum in these media are also included.

[0008] The medium for animal cells of the present invention is characterized in that a thymus extract is added to a basic medium. An animal thymus extract is an extract extracted from an animal thymus. These are pastes and powders that have been concentrated and dried. Here, the animal refers to domestic animals in general, and preferably includes cattle, buffaloes, sheep, goats, pigs, and the like.

The thymus is one of the pharyngeal derivatives of vertebrates and is an organ involved in immune response. The thymus is an organ that degenerates with aging, and is small in cows, less than 1 kg before maturity.

In the present invention, in order to prepare a thymus extract, not only raw thymus collected from an animal, but also frozen thymus, defatted thymus, dried thymus and the like prepared from these can be used. The thymus extract is obtained by washing the thymus collected from the animal in fresh water or diluted saline, washing with a homogenizer together with water, a salt solution and a buffer, and filtering with a filter press or the like to obtain a thymus extract. This thymus extract can be used after filtering through a microfilter to remove microorganisms.

The thymus extract may be obtained by pulverizing the thymus and then defatting or not defatting, or extracting the thymus with water or various salt solutions from a dry powder. The extract may further be heat-treated to remove heat-coagulable proteins. The thymus extract obtained in this way can be used as it is in the medium,
Further, after concentration or dry powdering, it can be used for various media. The percentage of thymus extract added to the basal medium is
Although not limited, in the case of thymus extract, the concentration is 20% or less as in the case of fetal calf serum. it can.

To produce the medium for animal cells of the present invention,
The thymus extract may be added to the basal medium at an arbitrary ratio. When the thymus extract is added to the medium, the nutritional balance is better for the animal cells, or the growth effect of the animal cells is equal to or higher than that of the basal medium supplemented with fetal calf serum. Therefore, even if the amount of fetal calf serum used in the basal medium is reduced, by adding the thymus extract,
The effect of the medium on the growth of animal cells can be brought closer to the basal medium containing fetal calf serum.

[0013]

The present invention will be described in more detail with reference to Test Examples and Examples. Test Example 1 <Preparation of various thymus test solutions> Extract from calf thymus acetone powder and extract from acetone-defatted calf thymus freeze-dried powder: powder obtained by defatting calf thymus acetone powder and calf thymus freeze-dried powder with acetone, 1g each in PBS
-(Ca, Mg free Dulbecco's phosphate buffer) well dispersed in 20 ml. Further, after sonication for 10 minutes, 3000rpm
The supernatant was obtained by centrifugation for 15 minutes at 0.2 μl.
The solution was filtered through a m-pore membrane filter.

F-3 solution: The supernatant was extracted from a raw calf thymus using a physiological saline solution, and then heat-coagulated protein was removed by centrifugation to obtain a supernatant. Acetone was added to this solution to a concentration of 90%, and the precipitate (F-
3) was obtained. 20 mg of this F-3 was dispersed in 10 ml of PBS, sonicated for 10 minutes, and then filtered through a 0.2 μm pore membrane filter to prepare a solution. Thymus extract dry powder (LMT) solution: Lyophilized calf thymus was defatted with acetone, extracted with water, centrifuged, and the supernatant was heated until the liquid temperature reached 80 ° C., followed by centrifugation (3,000
(rpm, 10 minutes) to remove the protein coagulated by heating to obtain a supernatant. This was spray-dried to obtain a thymus extract dry powder (LMT). 5 g of this LMT was added to 100 ml of PBS-
The supernatant obtained by autoclaving and centrifuging at 3000 rpm for 15 minutes was filtered through a 0.2 μm pore membrane filter to obtain an LMT solution.

<Preparation of Medium> A medium obtained by adding 1% of fetal bovine serum (FBS) to a DMEM medium is used as a basic medium, and the medium of Table 1 containing 0.01% of a test sample is prepared using the above test sample solution. did. <Culture of animal cells> Transfer each medium to a 96-well microplate.
Dispense 75 μl into 1 × 10 ⁵ cells / ml 18 PDL (popula
75 ul of bovine aortic endothelial cell (BAE) suspension (action doubling level: the number of cell population divisions) was added, the plate was sealed with vinyl tape, and the plate was cultured in a carbon dioxide incubator (5% CO ₂ concentration) at 37 ° C. . Culture 48, 72
After time, neutral red (NR) is dissolved in 20 mM HEPES to a concentration of 0.05%, and then 1% equivalent of FB
S was added, and 75 μl of an NR solution filtered through a membrane filter having a pore size of 0.2 μm was added.
Was incorporated into the cells. After gently aspirating and removing the culture solution containing NR, 300 μl of PBS was gently added, and this was gently aspirated off to wash the cells. 50% ethanol
After adding 200 μl of a phosphate solution and allowing to stand for 1 hour, the absorbance at 546 nm was measured. Table 2 shows the test results.

[0016]

[Table 1] As a result of the test, the uptake of NR at 48 hours of culture was higher than that in the control group (DMEM medium + 1% FBS).
Was large. Even after 72 hours of culturing, the value of the extract to which the extract from acetone powder or acetone defatted powder was added was higher than that of the control. Since the amount of NR uptake is proportional to the degree of cell growth, it was found that culturing BAE in a medium supplemented with thymus acetone powder, an extract of acetone-defatted thymus powder, and LMT promoted the growth. Was.

[0017]

[Table 2] Example 2 Calf freeze-dried powdered thymus was defatted with acetone and extracted with water to obtain an extract. After the extract was heated until the temperature of the extract reached 80 ° C., centrifugation (3,000 rpm, 10 minutes) was performed to remove the heat-coagulated protein to obtain a supernatant. This was spray-dried to obtain a thymus extract dry powder (LMT). Next, ME
This LM is added to a basic medium obtained by adding 0 to 5% of FBS to the M medium.
The medium of Table 3 to which T and 0.01% were added was prepared. 75 μl of each medium was dispensed into a 96-well microplate, and 75 μl of bovine aortic endothelial cell (BAE) suspension (1 × 10 ⁵ cells / ml) with 18 cell population divisions was added thereto. (5% CO ₂ concentration). After culturing for 3, 24, 48, and 72 hours, the NR solution was 75 μm as in Test Example 1.
l was added and the cells were cultured for an additional hour to incorporate NR into the cells. After gently aspirating and removing the culture solution containing NR, P
The cells were washed by gently aspirating and removing 300 μl of BS- gently. 50% ethanol / phosphate solution 200μl
, And allowed to stand for 1 hour.
Table 3 shows the results measured by the absorbance at 546 nm.

[0018]

[Table 3] In the serum culture in the presence of 0 to 5% FBS, NR uptake in the 0.01% LMT-added group was better than that in the absence of LMT, indicating that LMT promotes the proliferation of BAE cells. In this test, the efficacy of LMT on BAE growth was confirmed. However, when the amount of LMT was increased to 0.05%, almost no NR uptake was observed.

Example 3 A medium shown in Table 4 was prepared using a DMEM medium as a basic medium. This medium was placed in a 3.5 cmφ petri dish, 35 PDL human fetal lung fibroblasts (WI-38) were seeded at 2 × 10 ⁴ cells, and cultured in a 37 ° C. carbon dioxide incubator. At 24, 48, 72, 120 and 168 hours after the culture, the cells were recovered by trypsin treatment, stained with trypan blue, and the number of viable cells was counted.

[Table 4]

[Table 5] When cultured in a medium supplemented with 0.001% LMT as compared to the control, the number of viable cells was always higher after 24 hours. LM
In the group in which T0.01% was added, the number of cells remained the same as that in the control group up to the 120th hour of culture, but the number of viable cells decreased until 168 hours.

Example 4 A medium shown in Table 6 was prepared using a DMEM medium as a basic medium. Floating human fetal lung fibroblasts (WI-38) in each medium,
200 μl of this cell suspension was added to a 96-well microplate to give 2 × 10 ³ cells / plate, and after 24, 48, 72, 120 and 168 hours of culturing, the uptake of NR was observed. The results are shown in Table 7. The uptake of NR in cells cultured in a medium supplemented with 0.001% LMT was significantly increased at 120 and 168 hours of culture. In a medium supplemented with 0.01% LMT, the culture
Up to the 0th hour, NR uptake was equivalent to that of the control group, but decreased at the 168th hour of culture.

[Table 6]

[Table 7]

──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Yoshiaki Akabane 14 Nishinoshomonguchicho, Kichijoin, Minami-ku, Kyoto Nippon Shinyaku Co., Ltd. (72) Inventor Hidetaka Fuchu 21-3 Midoricho, Takatsuki-shi, Osaka Marudai Food Co., Ltd. (72) Inventor Yutaka Minegishi 21-3 Midoricho, Takatsuki-shi, Osaka Marudai Foods Co., Ltd. (72) Inventor Kyoden Yasumoto 34 Inspector 34-34, Iwakura-nakazai-cho, Sakyo-ku, Kyoto Minako Moto (56) References JP-A-60-137284 (JP, A) JP-A-64-90199 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) C12N 5/00 WPI (DIALOG)

Claims (1)

(57) [Claims] 1. A medium for animal cells characterized by adding an animal thymus extract to a basic medium.