JPS6384489A - Capsule for cultivation - Google Patents

Abstract

PURPOSE:To multiply cells, microorganisms, etc., in capsules with a high efficiency by improved cellular adhesive property, oxygen permeation rate, etc., by preparing the capsules for cultivation from a phosphazene based polymer having repeating units expressed by a specific formula. CONSTITUTION:Dichlorophosphazene trimer is thermally polymerized to give dichlorophosphazene polymer, which is then reacted with a reactive substance containing groups expressed by R<1> and R<2> in formula I to synthesize a phosphazene based polymer expressed by formula I. The above-mentioned polymer is formed into capsules 1, 3 or 5 covering a cultivation object liquid containing dispersed cells or microorganism which are cultivation objects in the interior thereof by a double-tube nozzle method, double emulsion method, etc. The polymerization degree of the phosphazene based polymer is preferably about 10<2>-10<4> and the thickness of the capsule wall of the capsules for cultivation is about 1-200mum.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a culture capsule used for enclosing and culturing animal and plant cells and microorganisms.

(Prior Art) In recent years, efforts have been made to cultivate animal and plant cells and microorganisms and obtain products from their metabolic activities.In particular, substances that are difficult to synthesize directly artificially, such as interferon, The production of hormones, lymphocytes, antibodies, enzymes, antibiotics, vaccines, etc., as well as the production of alcohols, organic acids, amino acids, etc. through the activities of microorganisms have become important technologies in the field of biotechnology. There is.

As a method for culturing cells and microorganisms, a culture solution and cells or microorganisms are placed in hollow particles made of synthetic or natural polymers, the hollow particles are placed in the culture solution, and the cells and microorganisms are grown in the internal space of the hollow particles. Culturing methods have recently begun to be considered.

In methods for culturing cells and microorganisms that utilize the internal space of hollow particles (hereinafter referred to as "capsules"), it is easy to separate the cells and microorganisms from the culture medium containing the products, and A major advantage is that the microorganisms are kept protected by the capsule, so the cells and microorganisms are not damaged even if the capsule collides with them.

However, the capsules used for culturing have the ability to pass through nutrients and oxygen necessary for the growth and survival of cells and microorganisms.
In addition, it is required to be made of a material that has the ability to permeate metabolic waste products such as carbon dioxide gas, ammonia, and lactic acid, and in some cases, the ability to permeate products. In addition, such culture capsules must not be toxic to cells or microorganisms, and must not be toxic to cells or microorganisms (adhesion-dependent cells). The material must have good adhesion and proliferation properties.

[Problem that the invention seeks to solve]

Hitherto, almost no culture capsules with such properties have been known, and only a few culture capsules made of hydrous gel of calcium alginate, polylysine, etc. are known.

However, these culture capsules have very low mechanical strength, and there is a high risk that cells and microorganisms sealed inside may leak out.

An object of the present invention is to provide a culture capsule that has various characteristics required for a culture capsule as described above and has high mechanical strength.

[Means for solving problems]

The culture capsule of the present invention is characterized by being made of a phosphazene-based polymer having a repeating unit represented by the following general formula.

General formula [However, R1 and R2 are the same or different, O
R3, NR'Rs again 4;! SiR'z C;-this 1?
, R3 is an alkyl group, an alkenyl group, a phenyl group, an alkylphenyl group, a halogenated group thereof, or a sugar residue, and R4 is a hydrogen atom, an alkyl group, a hydroxyalkyl group, or a phenyl group. When it is a group other than a hydrogen atom, it is an alkyl group, a hydroxyalkyl group, or a phenyl group; when R4 is a hydrogen atom, it is an amino acid residue, a polypeptide residue, or a glucosamine residue; and R6 is an alkyl group. or a halogenated alkyl group), and n represents the degree of polymerization. ]
The present invention will be specifically explained below.

In the present invention, a phosphazene polymer having a repeating unit represented by the above general formula is used as a material constituting a culture capsule for enclosing and culturing animal, plant, or microorganism cells.

In the general formula, R1 and R1 are the same or different, and are OR'', N R' RS Mataha S i R'
x, and examples of the alkyl group in R3, R4, RS and R6 include methyl group, ethyl group, isobutyl group, neopentyl group, etc., and examples of the halogenated alkyl group include trifluoromethyl group, trifluoroethyl group, trifluoroethyl group, etc. Examples of halogenated phenyl groups such as fluoropropyl group, heptafluoropentyl group, and octafluorohexyl group include chlorophenyl group, fluorophenyl group, and dichlorophenyl group; and examples of alkylphenyl groups include tolyl group, xylyl group, and nonylphenyl group. Examples of alkenyl groups include allyl group,
Examples of hydroxyalkyl groups such as isopentenyl group include hydroxyethyl group, hydroxypropyl group,
Examples of sugar residues such as hydroxyethoxyethyl group include residues of monosaccharides or polysaccharides such as glucose, maltose, fructose, mannose, dextran, and starch; examples of amino acid residues include glycine, alanine, phenylalanine, aspartic acid, Examples of the polypeptide residue include amino acid residues such as glutamic acid, and residues of multimers of the above-mentioned amino acids.

Among these, phosphazene-based polymers having OR3 in which R3 is a halogenated alkyl group, phenyl group, or alkylphenyl group are particularly preferred, and culture capsules formed from such phosphazene-based polymers are a source of nutrients, oxygen, and metabolic waste. Permeability of substances such as objects, products, etc.
It has excellent adhesion and growth properties for adhesion-dependent cells, and allows cells and microorganisms to be cultured with high efficiency.

Further, the phosphazene-based polymer used in the present invention may include a plurality of types of repeating positions in which the side chains (R' or R'') are different in the general formula.

The degree of polymerization of the phosphazene polymer used in the present invention is preferably 102 to 104.

When this degree of polymerization is too low, film forming properties are poor, making it difficult to form capsules, while when the degree of polymerization is too high, the resulting phosphazene polymer is difficult to dissolve, making it difficult to process it into capsules. This makes it difficult to form capsules that are thin and have a uniform thickness.

The intrinsic viscosity of the phosphazene polymer having the above polymerization degree is usually 0.5 to 3 dl/g.

As a method for synthesizing the phosphazene polymer represented by the above general formula, for example, a dichlorophosphazene trimer C/α is thermally polymerized at 150 to 350°C under vacuum to obtain a dichlorophosphazene polymer, and then this dichlorophosphazene polymer is obtained. A chlorphosphazene polymer and a reactive substance containing the groups represented by R1 and R2, such as sodium alkoxide, sodium phenoxide, alkylamine, sugar, amino acid, etc., are combined with diisopropyl ether, tetrahydrofuran, benzene, toluene, chlorobenzene, etc. By reacting in the presence of a solvent such as methylene chloride, chloroform, trichloroethane, acetone, methyl ethyl ketone, or cyclohexanone, the chlorine atom (C/) in the side chain of the above polymer is removed from the group represented by R1 and R2, such as an alkoxy group, phenoxy Examples include a method of substituting with a group, an amide group, etc.

Another method is to add the above dichlorophosphazene trimer and a reactive substance containing the groups represented by R1 and R2 to a solvent and react them.

The reactive substance containing the groups represented by R1 and R2 is
It can be appropriately selected depending on the type of cells and microorganisms to be cultured.

An example of a specific form of the culture capsule of the present invention is shown in FIG. 1. FIG. 5mm
It is considered to be a degree. These culture capsules are, for example, two semi-culture capsules IA each having a cylindrical shape with a bottom that fit into each other, each manufactured by an immersion process.

1B are combined. The total length of the combined culture capsule 1 is usually 5 to 30 mm, preferably 10 to 20 mm, and the film thickness is about 50 to 20 mm.

FIG. 1(b) shows a spherical culture capsule 3 having a single culture space 2 inside. This spherical culture capsule 3 is conveniently manufactured by, for example, a double tube nozzle method ("Microcapsule" Sankyo Publishing Co., Ltd. 977, etc.). That is, as shown in FIG. 2, it consists of an inner tube 12 having a circular opening 11, and an outer tube 14 which is coaxially arranged so as to surround this inner tube 12 and has an annular opening 13 formed by the inner tube 12. A double-front nozzle 10 is prepared, and the inner tube 12 is supplied with a culture solution in which cells or microorganisms to be cultured are dispersed, and the outer tube 14 is filled with a capsule-forming solution made of a phosphazene polymer solution. By supplying the capsule and applying constant vibration to the nozzle, a double droplet with a diameter of 0.1 to 5 mm is generated in which the droplet to be cultured is covered with the capsule forming liquid. Then, by dropping these double droplets into a non-solvent of the phosphazene polymer related to the capsule-forming liquid, such as paraffins such as petroleum ether, liquid paraffin, petroleum benzine, and ligroin, or water, cells or microorganisms are removed. A spherical culture capsule containing the dispersed culture target liquid can be obtained.

FIG. 1(c) shows a spherical culture capsule 5 having a plurality of vesicles 4 inside. This spherical culture capsule 5 is manufactured, for example, by a double emulsion method. As a specific example, a culture target liquid consisting of a dispersion of cells or microorganisms and a phosphazene polymer are mixed in a hydrophobic solvent such as diisopropyl ether, tetrahydrofuran, benzene, toluene, chlorobenzene, methylene chloride, chloroform, or trichloroethane. By forming an emulsion with the dissolved capsule-forming liquid, forming a double emulsion with this emulsion and an aqueous medium, and volatilizing the solvent of the phosphazene-based polymer, the phosphazene-based polymer is produced while containing the culture target liquid. A method of precipitation can be mentioned. It is preferable that the diameter of the particles obtained by this method is about 0.5 to 5 ml11, and the diameter of each of the internal vesicles 4 is 100 to 300 μ.

In the production of capsules by this method, vesicles 4
In some cases, the number of capsules becomes 1, resulting in a single capsule similar to the spherical culture capsule 3.

As the solvent for the phosphazene polymer used to prepare the capsule-forming liquid, for example, the same solvent as used in the synthesis of the phosphazene polymer (ethanol, water, etc.) can be used.

In the preparation of capsules, a crosslinking agent can be used as necessary, and such crosslinking agents include, for example, monosaccharides,
Examples include polyamines, peroxides, polyamines such as ethylenediamine, hexamethylenediamine, diethylenetriamine, polyethyleneimine, chitosan, and others.

The thickness of the capsule wall of the culture capsule of the present invention is appropriately selected depending on the cells or microorganisms to be cultured.
~200n.

The culture capsule of the present invention has excellent properties, and it is thought that the main reason for this effect is the high oxygen permeability of the capsule wall due to the phosphazene polymer. That is, when the culture capsule of the present invention encapsulating cells or microorganisms to be cultured is suspended or immersed in a medium, oxygen dissolved in the medium is transferred to the capsule wall of the culture capsule with high efficiency. Oxygen diffuses through the capsule wall, thereby supplying sufficient oxygen to the cells and microorganisms that are attached or floating inside the culture capsule, resulting in sufficient activity of the cells and microorganisms. This is thought to be because it can support the

Cells and microorganisms that can be cultured using the culture capsule of the present invention are not limited in any way; for example,
Human uterine cancer cells (HeLa), Chinese hamster lung cells (V-79), human fetal lung cells (MR(nee5)), chimpanzee liver fibroblasts, human foreskin cells, chicken fetal fibroblasts, primary monkey kidney cells , various adhesion-dependent cells such as mouse metastatic fibroblasts, pituitary tumor cells, African green monkey kidney cells (Vero), adrenal gland tumor cells, fused cells of myeloma cells and lymphocytes, leukocytes,
Planktonic cells such as lymphocytes, yeasts such as beer yeast, wine yeast, baker's yeast, soy sauce yeast, and feed yeast, Nocardi 7, actinomycetes such as Streptomyces, Escherichia coli, Bacillus subtilis, lactic acid bacteria, blue mold, Aspergillus aspergillus, etc. Examples include microorganisms such as ascomycetes, basidiomycetes such as shiitake mushrooms, nameko mushrooms, and enokitake mushrooms.

When culturing cells, as mentioned above, when a culture capsule containing a cell suspension is gently stirred in a medium, the cells either adhere to the inner wall of the culture capsule or proliferate in a suspended state. Therefore, by continuing the culture, metabolites are produced depending on the cell type, and the cells themselves can be obtained in large quantities.

The culture medium used for microorganisms includes carbon sources, nitrogen sources, inorganic salts, vitamins, etc., such as glucose, fructose, maltose, sucrose, starch, dextrin, blackstrap molasses, citric acid, fumaric acid, and ammonium sulfate. , ammonium chloride, ammonium phosphate, sodium nitrate, urea, amino acids, peptone, soybean whey, yeast extract, malt extract, meat extract, corn stable liquor, magnesium salt, phosphate, vitamin B+1 vitamin B1, vitamin C, etc. For cells, commercially available M
EM medium, DEM medium, etc. are used, and serum is generally added to the cells at a rate of about 10%.
In recent years, serum-free media have also become available.

When culturing microorganisms, in addition to the method described above, by filling a column with a culture capsule containing microorganisms and flowing a culture medium through this column, the microorganisms are multiplied and produced. By taking advantage of the fact that the product is eluted into the culture medium through the capsule wall, the product can be obtained in a state separated from the microorganism.

〔Example〕

Examples of the present invention will be described below, but the present invention is not limited to these Examples.

Example 1 (Manufacture of phosphazene-based polymer) 10 g of hexachlorocyclotriphosphazene, which had been sufficiently vacuum-dried, was polymerized for about 48 hours while maintaining the temperature at 210° C. using diethylaluminium ethoxide as a catalyst.

Next, the obtained polymer was dissolved in dehydrated benzene, and insoluble materials were filtered off using a glass filter.

The obtained filtrate was dropped into a tetrahydrofuran solution containing 1 g of sodium methoxide and 4 g of sodium phenoxide, and after the dropwise addition was completed, the reaction was carried out at 65° C. for 25 hours.

Note that the operations up to this point were performed under a nitrogen stream.

The resulting reaction product was neutralized, precipitated with ethanol, and dried to obtain 5.5 g of a pale yellow phosphazene polymer having methoxy and phenoxy groups. This is referred to as "phosphazene polymer A."

The intrinsic viscosity of this phosphazene polymer A in tetrahydrofuran was 1.02 dl/g.

(Manufacture of Capsules) 2 g of the above phosphazene polymer A was placed in an Erlenmeyer flask, and 40 g of diisopropyl ether was added and stirred to prepare a capsule forming liquid.

This is referred to as "capsule forming liquid 1."

Then, a stainless steel rod with a diameter of 31 mm and a hemispherical tip was heated to about 80°C, immersed in capsule forming solution 1 to a depth of 1 cffi, pulled up, and then immersed in ethanol. The thickness is about 20 trm at the tip of
A slightly hard membrane was formed, and this was pulled out from the rod to form a cylindrical container to obtain a semi-culture capsule.

A set of two semi-culture capsules obtained in this way was prepared, and 10,000 lactic acid bacteria were added to the medium in one semi-culture capsule/d.
A culture capsule containing lactic acid bacteria was produced by adding a culture solution dispersed at a concentration of 1 and capping it with the other semi-culture capsule.

(Culture and production of lactic acid) Using a glucose solution containing the following components at a concentration of 10% by weight as a medium, place 11 into a culture container, add 100 culture capsules containing the lactic acid bacteria obtained as described above, and stir. Culture was performed at a speed of 15 r, p, ra, and a temperature of 30°C. The dissolved oxygen concentration of the medium during culture is 6~
10 pp steel, pH kept at 4.7, medium at 100 d/
Exchanged at a rate of time.

■KffiHPOa for the bottom of the burial mound 1 g/fiM
g S Oa・7 Hz 0 200 mg/
lFe5Os・IHzo 10 mg/
RCaC1, 10mg/I Total amount of inorganic salts of Mn, MO, CI+, , CO and Zn 0.02-0.5mg/e
After 4 hours had elapsed, the culture capsule was taken out and the concentration of lactic acid bacteria was examined, and it was found to be 2.5 million cells/d, and after 2 days had elapsed, the culture capsule was filled with lactic acid bacteria.

100 culture capsules filled with lactic acid bacteria were packed into a column with an inner diameter of 20+ am, and the culture medium was poured from below at 10-
When lactic acid was separated by flowing the column at a rate of 7 hours, the proportion of lactic acid in the medium that passed through the column was 0.6 g/N-hr under conditions of a residence time of about 12 hours.

Example 2 (Production of phosphazene polymer) A solution obtained by dissolving the polymer log obtained from hexachlorocyclotriphosphazene in dehydrated benzene in the same manner as in Example 1 was first dissolved in di-n-butylamine 5
The mixture was added dropwise to a tetrahydrofuran solution in which 10 gtt of sodium phenoxide was dissolved and reacted. The operations up to this point were performed under a nitrogen gas flow.

The obtained reaction product was neutralized, precipitated with ethanol, and dried to obtain 8.5 g of an elastic phosphazene polymer having dibutylamino groups and phenoxy groups. This is referred to as "phosphazene polymer B."

The intrinsic viscosity of this phosphazene polymer B in tetrahydrofuran was 1.5 di/g.

(Manufacture of Capsules) A capsule-forming liquid was prepared by dissolving 2 g of this phosphazene-based polymer B in 50 parts of chlorobenzene. this"
"Capsule Forming Liquid 2".

Separately, Chinese hamster lung fibroblast rV-7
9 cells j were dispersed in MEM medium containing fetal bovine serum (Fe2) at a ratio of 10% by volume, and the number of cells was adjusted to 4 x 10' cells/17 to obtain a culture solution.

It has the configuration shown in FIG. 2, the diameter r of the circular opening 11 of the inner tube 12 is 5On, and the width d of the annular opening 13 is 2O.
A dispersion of ■-79 cells is encapsulated by supplying the culture target liquid to the inner tube of the double-tube nozzle of n, and supplying capsule-forming liquid 2 to the outer tube, and dropping it drop by drop into petroleum ether. The outer diameter of the phosphazene polymer B is approximately 1.
A 5 m+m culture capsule was formed, collected and washed with MEM medium, and then transferred to MEM medium containing Fe2 at a ratio of 10% by volume. The capsule wall thickness of this culture capsule was about 1 On.

(Culture of cells) Approximately 100 tZ (sediment volume) of the culture capsules obtained in this manner were added to a volume of 1J! into a spinner flask. Then, using MEM medium containing 10% by volume of Fe2 as a medium, stir gently at a temperature of 35°C (10 to 15%
r, p, m,), and the dissolved oxygen concentration is 8PPIIl, p
Culture was performed while keeping H at 7.

After culturing for 72 hours, the culture capsule was collected and destroyed, and it was found that the cells adhered to the inner wall of the culture capsule and were proliferating. When the cells were detached with trypsin and counted, it was calculated that the cells had proliferated to 90 times the original number. Furthermore, no cells were observed in the medium outside the culture capsule or in the outer wall of the culture capsule after the completion of the culture, and rV-
There was no leakage of 79 cells.

〔Effect of the invention〕

As can be understood from the above explanation, since the culture capsule of the present invention is made of a phosphazene polymer, it is nontoxic to animal, plant, and microorganism cells, and has excellent adhesion to adhesion-dependent cells. Moreover, the oxygen permeation rate of the culture capsule is high, and the oxygen in the culture medium efficiently diffuses into the culture capsule, and as a result, the cells and microorganisms inside the culture capsule receive a sufficient supply of oxygen and have a high oxygen permeation rate. Can multiply efficiently.

In addition, in the formation of phosphazene polymers, there is a wide selection range of groups that can be introduced as side chains, so depending on the type of adhesion-dependent cells to be cultured, it is possible to select the optimal side chain for the cells. As a result, it is possible to obtain a culture capsule having optimal characteristics according to the cells to be cultured.

[Brief explanation of the drawing]

Figures 1 (a) to (c) are explanatory cross-sectional views showing specific examples of the culture capsule of the present invention, and Figure 2 (a)
and (b) are an explanatory cross-sectional view and an end view of the tip portion of the double front nozzle. 1...Culture capsule IA, IB...Semi-culture capsule 2...Culture space 3.5...Spherical culture capsule 4...Vesicle 10...Double front nozzle 11
．． 13...Opening 12...Inner tube 14...Outer tube Takashi Figure 1 (A) (B)
()\)Article 2 Figure

Claims (1)

[Scope of Claims] 1) A culture capsule characterized by being made of a phosphazene polymer having a repeating unit represented by the following general formula. General formula ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ [However, R^1 and R^2 are the same or different, OR^3, NR^4R^5 or SiRa^6_3 (
Here, R^3 is an alkyl group, an alkenyl group, a phenyl group, an alkylphenyl group, a halogenated group thereof, or a sugar residue, and R^4 is a hydrogen atom, an alkyl group, a hydroxyalkyl group, or a phenyl group. Yes, R^5 is an alkyl group, hydroxyalkyl group, or phenyl group when R^4 is a group other than a hydrogen atom, and R^5 is an alkyl group, hydroxyalkyl group, or phenyl group.
When 4 is a hydrogen atom, it is an amino acid residue, a polypeptide residue, or a glucosamine residue, and R^6 is an alkyl group or a halogenated alkyl group), and n
represents the degree of polymerization. ]