JPS62179384A - Production of gel for cell culture - Google Patents

Abstract

PURPOSE:To obtain the titled gel which requires no strict temperature control in preparation of a gelatinous medium (solid medium) for cell culture and has simple operation, by gelatinizing an aqueous solution of spirulinan in the presence of a cationic ion. CONSTITUTION:An aqueous solution of spirulinan is preferably blended with a liquid medium for animal cell culture preferably containing a cation (preferably K<+>, Ca<++> or NH4<+>) in the presence of the cation and gelatinized preferably at room temperature to give the aimed gel.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a method for producing a cell culture medium using spirulinan.

[Conventional technology]

Agar culture methods are widely used in animal and plant tissue culture, cell culture, virology, bacteriology, etc., for isolation of pure single clones, long-term preservation of cells and bacteria, and detection of transformation.

Agar culture methods for colony formation of animal cells include a soft agar colony formation method and a plate agar culture method. The former consists of two layers: a support layer and a cell seeding layer, and the support layer usually contains 0.
5% agar is used and 0.33-0.4% agar is used for the cell seeding layer. This method is widely used for detecting transformants and forming animal cell colonies. The latter consists of IN agar, usually 0.5% (for animal cell colony formation) or 1.2-1.5% (for microbial preservation and colony formation, for plant tissue culture). In the soft agar method for culturing animal cells, seedlings are sterilized using agar under high pressure, then cooled to 40-43°C in a constant temperature bath, mixed with an equal volume of twice the concentration medium, and then added with serum to a concentration of 10%. Add to this and let it solidify to create a support layer. To create a cell seeding layer,
Mix 0.5% agar medium and cell suspension at a ratio of 2:1, and dispense this 0.33% agar cell suspension onto the support layer. Agar for the flat plate method is prepared in exactly the same way as the support layer for the soft agar method.

The above-mentioned agar culture method has the disadvantage that the agar solidifies at temperatures below 40 to 45°C. In particular, in the soft agar method, when mixing 0.5% agar medium and cell suspension, the former is
It is necessary to keep the temperature between 0 and 42°C, the latter at 37°C, and quickly mix and dispense. If the temperature is too high, there is a risk of damaging the cells, and if the temperature is too low, the agar will solidify during the operation.

That is, strict temperature control is required, the operation is relatively complicated, and skill is required. The present invention solves these problems and provides a method for producing a cell culture medium with a high colony formation rate through simple operations.

Means for Solving Problem C] The above problem is solved by a method for producing a cell culture medium, which is characterized by gelling an aqueous solution of spirulinan in the presence of cations.

(Specific explanation) Spirulina used in the present invention is Spirulina (
It is a mucilage polysaccharide produced by algae of the genus Spirulina, such as Spirulina subsalsa 5ubsalsa), and its physicochemical properties and manufacturing method are described in detail in Japanese Patent Application No. 152147/1982. ,
An example of the manufacturing method is described in the Reference Examples of this specification. Spirulinan is divided into monospirulinan, which is insoluble in a 0.3 M aqueous potassium chloride solution, and λ-spirulinan, which is soluble in a 0.3 M aqueous potassium chloride solution. All of these are the same in that they gel in the presence of cations. Therefore, in this invention, one spirulinane, λ
- Both spirulinane and mixtures thereof can be used. In the present invention, these are simply referred to as spirulinanes.

According to the method of the present invention, any of animal cell culture media, plant cell culture media, and microbial culture media can be produced. However, since conventional agar media have various drawbacks in culturing animal cells, the advantages of the method of the present invention are particularly exhibited in the production of media for culturing animal cells.

In carrying out the invention of the present invention, first, a sterilized spirulinan aqueous solution and a highly concentrated cell culture medium are prepared separately, and then they are mixed uniformly and immediately placed in a culture container, such as a petri dish. There are 4 people inside. Although the mixing ratio (volume ratio) of the spirulinan aqueous solution and the cell culture medium is not critical,
It is convenient to set the ratio to approximately 1:1. This ratio is 1:1
In this case, the concentration of spirulinan in the aqueous spirulinan solution before mixing is conveniently about 1 to 3 w/v%, preferably about 2 w/v%. This concentration is I
If it is lower than W/V%, it becomes difficult to form a gel;
If it is higher than w/v%, it becomes difficult to prepare a solution of spirulinan. Since spirulinan itself is resistant to heat and easily dissolves in water at high temperatures, it is preferable to sterilize the spirulinan aqueous solution by heating according to the conventional method.
It is preferable to sterilize in an autoclave at 20° C. for 20 minutes.

When the mixing ratio is 1:1, the concentration of the cell culture medium before mixing is twice as high. The culture medium can be sterilized by any conventional sterilization method most suitable for the culture medium, such as autoclave sterilization or sterile filtration.

Inorganic cations such as K", Ca", and HN4+ are effective for gelation of spirulinan, and the concentration required for gelation is:
For example, for 1, it is approximately 0.01M or more. Since this level of cations is normally present in the cell culture medium, there is no need to specifically add cations for gelation. However, needless to say, if the medium itself does not contain sufficient cations for gelation of spirulinan, cations that do not affect the target cells may be added for the purpose of promoting gelation.

According to the method of the present invention, the spirulinan aqueous solution and the culture medium do not gel even at low temperatures unless the two are mixed. Therefore, both are kept at room temperature or culture temperature, e.g.
After cooling to 0.degree. C., cells can be inoculated into the medium, and then the two can be mixed to form a gel. Therefore, cells are not damaged by heat.

〔Effect of the invention〕

According to the method of the present invention, there is no need for strict temperature control when preparing a gel culture medium (solid medium) for cell culture.
Easy to operate. Furthermore, since the cells are not exposed to high temperatures due to the steel culture medium, there is little risk of damage to the cells due to high temperatures. Furthermore, spirulinan does not inhibit the growth of cells, and as a result of these factors, the spirulinan medium produced by the method of the present invention has better colony forming properties than a conventional agar medium.

Note that the above effects are particularly noticeable in animal cell culture.

Next, the method of the present invention will be specifically explained using examples.

1-type spirulinan was added to water to a concentration of 2%, dissolved in an autoclave (120° C., 20 minutes), and then cooled to room temperature. Next, double the concentration of commercially available RPMI 1
640 medium [containing 20% FC3 (fetal bovine serum)] 5ml
human lymphocyte cells (6-thioguanine resistant, 67G-
Suspend 5 x 10' cells of cell suspension #8) and immediately add 2.5 ml of this cell suspension medium to 2.5 mL of type 1 spirulinan aqueous solution.
mj! and poured into a 5 ml petri dish. This was cultured for 15 days in a 37°C incubator (5% CO! ventilation). The number of colonies produced was 260.

For comparison, 0.66% (w/v) agar was dissolved in an autoclave (120 °C, 20 min) and
It was kept warm in a water bath at ℃. Warm 2x concentrated PR old 1640 medium (containing 20% FC3) to 40°C, add 6TG-
WIL#8 cells were suspended (5×10” cells in 15 mj).

Add 2.5 mj2 of this cell suspension medium to an agar aqueous solution of 1.5 mj2.
ml at 40°C and poured into a 5 ml petri dish. 15 in a 37°C warmer (5% CO! ventilation)
After culturing for days, the number of colonies formed was 120.
It was.

1st branch five earth spirulinan 6 A medium having the following composition was prepared.

KzHPOnO,5i CaC1zO,04; 1l
zo 101; NaNO22,5; Fe50*
0-01; Macll, 0; EDTA
O, 08; (*) 5 solution H3BO32, 86g; MnCj! z・4f
hO1,81g 1ZnSOn・7HzOO,22;
CuSO4・5Hz0 0.08; Na, Mo
40.021; Conc-H, So, 1 drop
;H2O1,07! Spirulina subsalsa was inoculated into 100 ml of this medium and cultured at 37° C. to 40° C. under irradiation with a fluorescent lamp of 4000 lux and without aeration of carbon dioxide. It was confirmed that mucilage polysaccharide was secreted outside the algae during the culture process. Under these conditions, this strain grew by elongating filamentous algal cells and floated to the surface of the liquid. It formed a more compact spiral filament compared to Spirulina platensis.

Wash algae with water, add 80% methanol, 80% acetone, 1
The dye was removed by boiling with 0.00% ethanol and ether. The algal bodies were collected and the spirulinan was extracted by heating at 90° C. for 1 hour in an aqueous solution containing 0.2% sodium chloride and 0.1% sodium bicarbonate, and filtered.

Spirulinan was precipitated by adding cetyltrimethylammonium bromide (Cetabron) to this spirulinan extract at a concentration of 2%. This precipitate is 8
By washing with 0% ethanol, 100% ethanol, and ether and drying, 44 cm of white spirulinan was obtained.

Yutaka 11J2. λ-Spirulinan and Spirulina 0 produced according to the method described in Example 1
．． 1 g was heated to 90°C while stirring vigorously.
Dissolved in distilled water to a concentration of 1% and filtered. To the filtrate was added 0.1 volume of 3M potassium chloride solution, the solution was mixed and cooled in a crushed ice bath for 1 hour. Centrifugation at 10,000 rpm+s for 1 hour at 50°C;
It was divided into an insoluble fraction and a soluble fraction.

The insoluble fraction was resuspended in 0.3M sodium chloride solution,
Chlorine was removed by centrifugation and washing the insoluble matter with 80% warm ethanol, and 100% ethanol,
After washing with acetone and ether and drying, 0.03 g of monospirulinane was obtained.

On the other hand, the soluble fraction was precipitated by adding 2.5 volumes of 100% ethanol, washed with 80% ethanol, 100% ethanol, acetone, and ether, and dried;
0.069 g of λ-spirulinan was obtained.

The ratio of λ-spirulinan to -spirulinan is approximately 69
．． 1:30.9.

Claims (1)

[Scope of Claims] 1. A method for producing a cell culture medium, which comprises gelling an aqueous solution of spirulinan in the presence of cations. 2. Claim 1, characterized in that an aqueous solution of spirulinan and a liquid medium for animal cell culture containing cations are mixed and gelled to produce a medium for animal cell culture. the method of. 3. The cation is K^+, Ca^+, or NH_
4^+, or a combination thereof, according to any one of claims 1 or 2. 4. The method according to claim 1 or 2, wherein the gelation is performed at room temperature.