JP2827298B2 - Cell culture carrier - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a carrier for cell culture, and more particularly to a carrier used when culturing animal cells. More specifically, the present invention relates to a cell culture carrier suitable for attaching animal cells to a main culture carrier and performing high-density culture in a stationary state, a suspension state, or the like.

(Prior Art) In recent years, various culture methods have been developed and rapidly developed for culturing animal cells. One of them is a microcarrier method using a culture carrier.

Many of the currently known microcarriers are anion-exchange type microcarriers into which only positively chargeable chemical residues have been introduced. For example, Cytodex-1
(Pharmacia), Super Beads (Flow Labs), Domacell (Pfeifer & Langen), DE-52, D
Microcarriers such as E-53 (manufactured by Whatman) and MC-series (manufactured by Waitaki) are obtained by chemically bonding a diethylaminoethyl group to a polymer carrier such as Sephadex or cellulose.

Also, JP-A-63-71173, JP-A-63-226282 and JP-A-64-10979 disclose that a water-insoluble polymer particle having a (meth) acrylate ester as a constitutional unit can be positively charged. Microcarriers comprising target residues have been described. In addition, a biocarrier (manufactured by Bio-Rad) having a dimethylaminopropyl group chemically bonded to a polyacrylamide gel is also known. On the other hand, on the other hand, biosilon (manufactured by Nunc) and cytosphere (manufactured by Lux) in which a negative charge is introduced by subjecting a polystyrene surface to discharge treatment are also known.

(Problems to be Solved by the Invention) However, the conventional culture carrier has a drawback that cells are not sufficiently adherent or proliferative, and cells are easily detached even after long-term culture. Therefore, there has been a demand for a culture carrier that has good cell adhesion and growth properties, is difficult to peel off even in long-term culture, and can easily achieve high-density culture.

An object of the present invention is to provide a cell culture carrier capable of culturing animal cells, in particular, adhesion-dependent animal cells in high density and in large quantities.

(Means for Solving the Problems) As a result of intensive studies in view of the above problems, the present inventors have found that the cause of cell detachment is that the carrier has only a positive charge or only a negative charge. A chargeable chemical residue;
Furthermore, it has been found that not only the adhesion and proliferation of cells are improved by using a negatively charged chemical residue in combination with the carrier surface, but also that the cells are hardly detached even in a long-term culture. Reached.

That is, the gist of the present invention is that the support itself is made of a water-insoluble polymer having a polymerizable monomer as a structural unit, or the support surface is coated with the water-insoluble polymer, and the polymer is A cell culture carrier comprising a positively chargeable chemical residue and a negatively chargeable chemical residue.

 Hereinafter, the present invention will be described in detail.

As the polymerizable monomer used in the production of the cell culture carrier of the present invention, a polymer obtained by polymerization has a low nonspecific adsorption to proteins and the like, and can efficiently convert a target vaccine protein produced by animal cells. Hydrophilic vinyl monomer (ie, CL) that prevents cell growth and expansion due to the hydrophobic environment of the polymer.
Monomer with low OGP value: The CLOGP value is a value obtained by calculating the logarithmic value of the partition coefficient of the monomer in the solute water-1-octanol system using silicic acid, and its significance will be described later. ) Are preferred. Typically, (meth) acrylic acid ester or (meth) acrylamide is used.
Specifically, diethylene glycol (meth) acrylate, triethylene glycol (meth) acrylate, tetraethylene glycol (meth) acrylate, octaethylene glycol (meth) acrylate, polyethylene glycol (meth) acrylate, glycerol (meth) acrylate, glycidyl ( Meth) acrylates,
Examples thereof include (meth) acrylamide, methyl (meth) acrylamide, and the monomers described in Tables 1 and 2.

Monomers having no chemical residues that may be charged in order to the polymer hydrophilic, polyoxyethylenes [(-CH ₂ -C
H ₂ —O) _n ], n is preferably 2 or more.

If the polymer obtained by polymerization here is water-insoluble, a cross-linking agent component is not necessary, but elution of the soluble component from the polymer and the support or peeling of the polymer from the support when coated is performed. Considering this, it is preferable to add a crosslinking agent component.

As the crosslinking agent component, a polyfunctional vinyl monomer having copolymerizability with the above-mentioned monomer component and having as high a hydrophilicity as possible (low CLOGP value) is preferable. For example, diethylene glycol di (meth) acrylate, triethylene glycol di (meth) acrylate, tetraethylene glycol di (meth) acrylate, polyethylene glycol di (meth) acrylate, glycerol di (meth) acrylate
Acrylate, trimethylolpropane tri (meth) acrylate, ethylenebis (meth) acrylamide, triethylenebis (meth) acrylamide and the like can be mentioned. The crosslinker component is preferably used to minimize leakage of components of the support, but if the crosslinker component content is too high, it is secreted by proteins and cells in serum. There may be a problem that the bioactive proteins are adsorbed and cannot be efficiently recovered.
Therefore, the content of the crosslinking agent component is preferably 0.1 to 50% by weight, particularly preferably 1 to 20% by weight.

In the present invention, a diluent is preferably added to the monomer. Thereby, the inside of the support can be impregnated and can be uniformly coated. In addition, the obtained water-insoluble polymer can be given an appropriate flexibility.

In the present invention, the content of positively chargeable chemical residues in the culture solution is 0.5 to 3.0 meq / g of water-insoluble polymer, and the amount of negatively chargeable chemical residues is When the amount is 0.01 to 2.0 meq / g of the water-insoluble polymer, more preferable results are obtained.

When the amount of the positively chargeable chemical residue is less than the above range, the adhesion is poor, and when the amount is more than the above range, charge inhibition is observed. When the amount of the negatively chargeable chemical residue is less than the above range, the adhesiveness is poor, and when the amount is less than the above range, the adhesive growth is poor.

It is not only the amount of chemical residues that affects cell adhesion and proliferation, but also the CLOGP value of a monomer unit corresponding to a positively chargeable chemical residue in a polymer. In order for cells to attach and grow on the support surface, the CLOGP of the corresponding positively chargeable monomer units in the polymer
Preferably, the value is in the range -1.5 to +2.0. The CLOGP system in the present invention is based on Pocoma University (California) [FACOM 3.33 by T. Nishioka (Kyoto University)].
Program compiled into a version], and is defined as follows: CLOGP value is based on the solute water
It is a value obtained by calculating a logarithmic value of a partition coefficient in a 1-octanol system. This has made it possible to quantitatively express the hydrophobicity of a neutral molecule in terms of charge.
The CLOGP value of the positively chargeable monomer unit in the present invention is also determined by this method. Some of the positively chargeable chemical residues are considered to be in a dissociated state in the culture solution, but may be used in WSHu [Biotechnology and Bioengineering Vol. 29, 1155-1163 (198).
7)] et al. Report that the effect of net charge on growth rate at pH 7.20 is small or has no regularity. Therefore, the degree of hydrophobicity of the corresponding positively chargeable monomer unit in the polymer may be determined by considering the CLOGP value of the neutral molecule. CLOG of corresponding monomer units
If the P value is too small, the adhesion of the cells to the culture carrier will be poor. Conversely, if the CLOGP value is too large, the adhesion and proliferation of the cells will be poor. In addition, the biologically active protein secreted by the cells is adsorbed on the culture carrier.
Furthermore, regularity was observed between the CLOGP value of the monomer unit and its content. That is, it was also found that, in order to adhere and grow cells on a culture carrier, it is preferable to increase the content when the CLOGP value is low, and to decrease the content when the CLOGP value is high. A monomer unit having a positively chargeable chemical residue and having a CLOGP value of -1.5 to
Those in the range of +2.0 include those shown in Table-1 and Table-2. On the other hand, as a method for introducing a negative charge into the water-insoluble polymer on the surface of the support, a method using a monomer having a negatively chargeable chemical residue in a polymer monomer and a method using a chemical bond after polymerization are used. There are two ways. Specific examples of a monomer unit having a chemical residue capable of being negatively charged and having a CLOGP value in the range of -1.5 to +2.1 include methacrylic acid and β-methacryloyloxyethyl hydrogen succinate. , Β-methacryloyloxyethyl hydrogen phthalate, methacryloxyethyl phosphate, sulfopropyl methacrylate and the like, and are shown in Table-3.

Of course, polymerizable monomers not shown in Tables 1, 2, and 3 can be used as long as they do not exceed the scope of the claims of the present invention. In the tables, Me represents a methyl group and Et represents an ethyl group.

The following can be suitably used as the structural support of the cell culture carrier in the present invention. Petri dishes, T-flasks, multi trays, roller bottles, paper (cellulose filter paper, etc.), cloth, foamable polymer (urethane, ether,
Esters), porous ceramics (silica-based, alumina-based), metal carriers, metal foils, glass beads, foamed glass, sheets, wood, fibers, and the like. The shape and size of these supports greatly vary depending on the culture method and culture scale. In order to observe the cells attached to the support, an optically transparent material is preferable, but this is not an essential condition for culture.

Examples of the shape of these carriers include a nodular shape, a spherical shape, a film shape, a film roll shape, a porous film shape, and a rod shape. As the spherical body, as a microcarrier, JP-A-63-71173, JP-A-63-226282 and JP-A-64-1
It can be produced by the method described in No. 0979.
Further, the film-like carrier can be produced by the method described in JP-A-64-47372. Agglomerated carriers can also be obtained by cutting by a suitable method after bulk polymerization of the monomer.

On the other hand, the method of coating the surface of the support with a water-insoluble polymer can be performed, for example, according to the following procedure. A polymerization initiator is added to a monomer solution obtained by mixing a monomer, a crosslinking agent, and a diluent. The solution is impregnated or coated on a support and excess monomer solution is compressed and squeezed out or flushed with nitrogen to remove. Next, the monomer is polymerized under a nitrogen atmosphere. Here, when producing a fixed bed carrier such as ceramics or foamable polyurethane, it is preferable to cut the carrier to 10 cm or less as much as possible in order to coat uniformly. In the case of producing a fixed bed carrier adapted to an incubator, after cutting and performing the above operation, an excess monomer solution is removed by a centrifuge. If the culture carrier is small, it can be impregnated and coated after filling in the column. A point to be noted in the above manufacturing process is that it is necessary to prevent the formation of a liquid pool inside the carrier when removing the excess monomer solution.

The diluent added to the monomer component may be any as long as it dissolves the monomer and is inert to the functional group of the monomer component, i.e., methacryloyl group, hydroxyl group, glycidyl group, amide group, amino group, carboxyl group, etc. and the support. . Suitable diluents depend on the type of monomer used, but are usually benzene, toluene, cyclohexane, ethyl acetate, butyl acetate, ethanol, propanol, butanol, 1-hexanol, cyclohexanol, octanol, heptanol, ethylene glycol, Examples include acetone, methyl isobutyl ketone, cyclohexanone, dibutyl ether, tetrahydrofuran, 1,4-dioxane, chloroform, dimethyl sulfoxide, acetonitrile, and a mixed solvent of the above water-soluble organic solvent and water.

When a good solvent is added to the monomer as the diluent, the coated water-insoluble polymer becomes an optically transparent polymer.

The amount of the diluent added to all monomers is 0 to 10 times. If the diluent is added in excess, not only the polymer membrane becomes brittle, but also problems such as a decrease in polymerization yield occur.

Examples of the polymerization initiator used include the following. That is, peroxide polymerization initiators such as benzoyl peroxide, lauroyl peroxide, and acetyl peroxide, 2,2 '
Polymerization can be initiated by an azo-based polymerization initiator such as -azobis-2-cyclopropylpropionitrile, or a redox-based polymerization initiator such as hydrogen peroxide-Fe ²⁺ salt.

Here, the selection of the polymerization initiator greatly differs depending on the monomer, the crosslinking agent, the diluent, and the like. When the monomer solution is water-soluble, redox-based polymerization initiator, water-soluble azo-based polymerization initiator and ammonium peroxide, potassium peroxide-
N, N, N ', N'-tetraazoethylenediamine and the like are preferably used.

As for the polymerization temperature, it is generally preferable that the redox-based polymerization initiator and the peroxide-based polymerization initiator are carried out at 30 ° C to 100 ° C.

Also, when there is a negatively chargeable chemical residue in the polymerizable monomer, the obtained polymer itself can be a negatively chargeable chemical residue, and in the polymerizable monomer, When a positively chargeable chemical residue is present, the resulting polymer can be a positively chargeable chemical residue as it is.
However, when the polymer does not satisfy the above-mentioned positive or negative functional group content, a functional group which can be positively charged by a chemical reaction must be further introduced by a chemical bond. When the polymer contains a glycidyl group, it can be introduced by reaction with an amine, and in the case of a hydroxyl group, 2,3-dihydroxypropyl group, etc., imide carbonate is obtained by reaction with cyanogen bromide. Later, it can be introduced by reaction with a diamine. When a negatively chargeable chemical residue is introduced by a chemical bond, a carboxyl group can be introduced by reacting the terminal hydroxyl group with an acetic acid halide in a basic solution. The method of combining these is described in detail by Mosbach (Methods in Enzymology, Vols. 134, 135, 136). It is often difficult to estimate the amount of positive and negative charges that can be introduced into the carrier depending on the support. In that case, the polymer obtained after separately polymerizing the monomer on a glass petri dish can be recovered, and the charge amount can be estimated by titration. The charge amount of the culture carrier obtained by coating shown in the examples is a value obtained by the above method.

The method of culturing using the carrier for cell culture of the present invention varies depending on the scale of culture, the purpose of culturing, and the like, and examples thereof include the following methods. When a petri dish or a T-flask is used, a suspension culture method is used. When a fixed bed is used, a fixed bed culture method is used. When an air lift is used, a reflux culture method is used.

The input amount of the culture carrier varies depending on the culture method, and is generally preferably in the range of 5% to 100% with respect to the culture solution.

(Examples) Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited to the following examples as long as the gist of the present invention is not exceeded.

Example 1 A spherical cell culture carrier (microcarrier) was produced by the method described below.

A thermometer, a cooling tube, a nitrogen introducing tube, and a stirring blade were attached to a 300 ml four-necked flask, and 30 g of potassium chloride / dihydrate, 50 ml of a 3% polyvinyl alcohol aqueous solution, and 85 ml of ion-exchanged water were stirred therein. 1-hexanol into this
60 g, cyclohexanol 30 g, polyethylene glycol methacrylate (trade name: PE-350, manufactured by NOF Corporation) 12.4
g, glycidyl methacrylate 6.0 g, methacrylic acid 0.8
g, polyethylene glycol methacrylate (trade name: 4G
0.8 g, 2,2'-azobis- (2,4-dimethylvaleronitrile) (trade name: V-6) as a polymerization initiator
An organic solvent in which 0.02 g of 5 (Wako Pure Chemical Industries, Ltd.) was dissolved was added to the flask. The temperature was gradually raised, and a polymerization reaction was performed at 65 ° C. for 5 hours under a nitrogen atmosphere. After completion of the reaction, the polymer was placed on a Buchner funnel and washed with a 50% aqueous methanol solution.
The polymer was put back into the flask. This operation was repeated, and the resultant was washed four times until the odor of 1-hexanol and cyclohexanol disappeared. The obtained polymer was a colorless transparent sphere. The polymerization yield was 87%. The particle size of this polymer was adjusted to 120 μm to 130 μm using a standard sieve.

The following reaction was further performed using this polymer. 30ml
The polymer (in water) was thoroughly washed with 1,4-dioxane and replaced. This was put in a 300 ml flask, and 30 ml of dioxane was further added. 5.0 g of ethanolamine in this
Was added dropwise, and an amination reaction was performed at 70 ° C. for 4 hours. After the completion of the reaction, the polymer was sufficiently washed.

Based on elemental analysis and acid-base titration, the amine functional group content was 1.
46 meq / g, carboxylic acid functional group content is 0.26 meq / g
Met. The degree of swelling of the polymer in a phosphate buffer solution (hereinafter abbreviated as PBS) is 17.9 ml / g, and the average particle size is 184 μm.
m, specific gravity was 1.03 g / ml. The corresponding positively charged monomer unit is hydroxyethylamino methacrylate-
2-Hydroxyethyl whose CLOGP value is -0.669, and the corresponding negatively charged monomer unit is methacrylic acid, whose CLOGP value is 0.470.

(Example-2) A spherical cell culture carrier (microcarrier) was produced by the method described below.

A thermometer, a cooling tube, a nitrogen inlet tube, and a stirring blade were attached to a 300 ml four-necked flask, and 30 g of calcium chloride / dihydrate, 50 ml of a 3% polyvinyl alcohol aqueous solution, and 85 ml of ion-exchanged water were stirred therein. 30 g of 1-hexanol, 30 g of cyclohexanol and 30 of 1-octanol
g, polyethylene glycol methacrylate (trade name: PE
-200, manufactured by NOF Corporation) 12.7 g, glycidyl methacrylate 5.4 g, β-methacryloyloxyethyl hydrogen succinate (trade name: SA, manufactured by Shin-Nakamura Chemical Co., Ltd.) 1.1 g,
Glycerol dimethacrylate (trade name: NK Ester 70)
0.8 g of an organic solvent prepared by Shin-Nakamura Chemical Co., Ltd. and 0.02 g of 2,2'-azobis- (2,4-dimethylvaleronitrile) (V-65: described above) as a polymerization initiator are added to the flask. Was.
The temperature was gradually raised, and a polymerization reaction was carried out at 65 ° C. for 5 hours under a nitrogen atmosphere. Post-treatment after completion of the polymerization was performed in the same manner as in Example-1. The amination was performed in the same manner as in Example 1, except that 3-amino-1-propanol was used instead of ethanolamine.

Based on elemental analysis and acid-base titration, the amine functional group content was 1.
37 meq / g, carboxylic acid functional group content is 0.22 meq / g
Met. The degree of swelling of the polymer in a PBS solution is 18.
8 ml / g, the average particle size was 192 μm, and the specific gravity was 1.03 g / ml.
The corresponding positively charged monomer unit is hydroxypropylamino-2-hydroxypropyl methacrylate, which has a CLOGP value of -1.125, and the corresponding negatively charged monomer unit is β-methacryloyloxyethyl hydrogen succinate. And its CLOGP value is 0.866.

(Example-3) A spherical cell culture carrier (microcarrier) was produced by the method described below.

A thermometer, a cooling tube, a nitrogen inlet tube, and a stirring blade were attached to a 300 ml four-necked flask, and 30 g of calcium chloride / dihydrate, 50 ml of a 3% polyvinyl alcohol aqueous solution, and 85 ml of ion-exchanged water were stirred therein. 30 g of 1-hexanol, 60 g of cyclohexanol, 12.7 g of polyethylene glycol methacrylate (PE-350: described above), 4.4 g of dimethylaminoethyl methacrylate, β-methacryloyloxyethyl hydrogen succinate (SA: described above) 1.1
g, polyethylene glycol dimethacrylate (trade name:
0.8 g of NK ester 9G, manufactured by Shin-Nakamura Chemical Co., Ltd., 2,2'-azobis- (2,4-dimethylvaleronitrile) as a polymerization initiator
(V-65: described above) An organic solvent in which 0.02 g was dissolved was added into the flask. The temperature was gradually raised, and a polymerization reaction was carried out at 65 ° C. for 5 hours under a nitrogen atmosphere. The post-treatment after the completion of the polymerization is as described in Example-1.
The same was done. In Example-3, no amination was performed. From elemental analysis and acid-base titration, the amine functional group content was 1.23 meq / g and the carboxylic acid functional group content was 0.25
It was meq / g. The swelling degree of the polymer in the PBS solution is 17.3 ml / g, the average particle size is 181 μm, and the specific gravity is 1.03 g / ml.
Met. The corresponding positively charged monomer unit is dimethylaminoethyl methacrylate, whose CLOGP value is
0.290, and the corresponding negatively charged monomer unit is
β-methacryloyloxyethyl hydrogen phthalate and its CLOGP value is 2.1.

(Example-4) A film-shaped cell culture carrier was produced by the method described below.

Dissolved in 30 g of ethylene glycol, 12.4 g of polyethylene glycol methacrylate (PE-350: described above), 5.4 g of glycidyl methacrylate, 0.85 g of methacrylic acid, 0.8 g of polyethylene glycol dimethacrylate (4G: described above), and 0.5 ml of methylene chloride as a polymerization initiator 2,2'-azobis- (4-methoxy-2,4-valeronitrile) (trade name: V
-70, manufactured by Wako Pure Chemical Industries, Ltd.) to prepare a 0.02 g monomer solution.
Nitrogen gas was introduced into this solution to remove dissolved oxygen. 0.
The monomer solution was poured into a cell consisting of two glass plates with a 5 μm polyester film spacer scissors. Soak this glass plate in a 60 ° C water bath for 10 minutes,
The polymerization reaction was performed under a nitrogen atmosphere. After the completion of the polymerization, the film gel was taken out and washed with a 50% aqueous methanol solution.
The film gel was colorless and transparent.

The film gel having a surface area of 3,600 cm ² was sufficiently washed with dioxane and replaced with dioxane. This was put in a 100 ml flask, and further 30 ml of dioxane was added. Into this, 5 ml of a dioxane solution of 1.0 g of 2-aminoethanethiol
Was added dropwise to carry out an amination reaction at 70 ° C. for 4 hours. After the completion of the reaction, the polymer was sufficiently washed. From elemental analysis and acid-base titration, the amine functional group content was 1.53 meq / g and the carboxylic acid functional group content was 0.29 meq / g. Specific gravity is 1.03
g / ml. The thickness was about 1.0 μm.

The corresponding positively charged monomer unit is mercaptoethylamino-2-hydroxypropyl methacrylate and has a CLOGP value of 0.375, and the corresponding negatively charged monomer unit is methacrylic acid and has a CLOGP value of 0.470.
It is.

(Example-5) A film-shaped cell culture carrier was produced by the method described below. 26 g of 1,4-dioxane, glycerol methacrylate (trade name: Blemmer GLM, manufactured by NOF Corporation) 9.7
g, 3.4 g of acrylamide, 4.1 g of diethylaminoethyl methacrylate, 0.9 g of β-methacryloyloxyethyl hydrogen succinate (SA: described above), 0.8 g of glycerol dimethacrylate (701: described above), and 0.5 ml of methylene chloride as a polymerization initiator. A monomer solution consisting of 0.02 g of 2,2'-azobis- (4-methoxy-2,4-valeronitrile) (V-70: described above) was prepared. Nitrogen gas was introduced into this solution to remove dissolved oxygen. In a cell consisting of a glass plate with a 0.5μm polyester film spacer scissors,
The above monomer solution was poured. This glass plate was immersed in a water bath at 60 ° C. for 10 minutes and polymerized under a nitrogen atmosphere. After the polymerization, the film gel was taken out and washed with a 50% aqueous methanol solution. The obtained film gel was colorless and transparent.

Based on elemental analysis and acid-base titration, the amine functional group content was 1.
53 meq / g, carboxylic acid functional group content is 0.29 meq / g
Met. The specific gravity was 1.03 g / ml. The film thickness is about 1.0μm
Met. The corresponding positively charged monomer unit is diethylaminoethyl methacrylate, whose CLOGP value is
1.981, and the corresponding negatively charged monomer unit is
β-methacryloyloxyethyl hydrogen succinate and its CLOGP value is 0.866.

(Example-6) A cell culture carrier having a foamable polyurethane as a support was used.
It was manufactured by the method described below.

30 g of ethylene glycol, 12.4 g of polyethylene glycol methacrylate (PE-350: described above), 5.4 g of glycidyl methacrylate, 1.05 g of β-methacryloyloxyethyl hydrogen succinate, 0.8 g of polyethylene glycol dimethacrylate (4G: described above), as a polymerization initiator A monomer solution was prepared by dissolving 0.02 g of 2,2'-azobis- (4-methoxy-2,4-valeronitrile) (V-70: described above) in 0.5 ml of methylene chloride. Nitrogen gas was introduced into this solution to remove dissolved oxygen. Foamable polyurethane (average pore diameter 80 μm, semi-continuous foam, manufactured by MD Kaseisha) (10 cm
× 10 cm × 7 cm) impregnated with the monomer solution prepared above,
Excess monomer solution was compressed and squeezed out. The polyurethane pieces were placed in a glass container, heated to 60 ° C. under a nitrogen atmosphere, and a polymerization reaction was performed under a nitrogen atmosphere for 2 hours. After the end of the polymerization reaction, to remove residual monomers and dioxane, 50
Washing was carried out with an acetone solution heated to ° C. and finally with distilled water.

Further, after replacing the polyurethane piece with dioxane,
Into this, 5 g of a dioxane solution of 1.5 g of an ethanolamine solution
l was added dropwise and amination was carried out at 75 ° C. for 4 hours. After completion of the reaction, the polyurethane pieces were sufficiently washed with a 50% aqueous methanol solution, and then washed with distilled water. The amount of coated polymer was 8.7%.

Estimated amount of positively chargeable functional groups is about 1.45 meq
/ g, the amount of negatively chargeable functional groups is about 0.19 meq / g
It is. The surface area was 90 cm ² / ml and the porosity was about 87%. The corresponding positively charged monomer unit is hydroxyethylamino-2-hydroxypropyl methacrylate, which has a CLOGP value of -1.125, and the corresponding negatively charged monomer unit is β-methacryloyloxyethyl hydrogen succinate. And its CLOGP value is 0.866.

(Example-7) A cell culture carrier using a porous ceramic as a support was used.
It was manufactured by the method described below. 1-hexanol 22
g, polyethylene glycol methacrylate (trade name: PE
-90, manufactured by NOF CORPORATION) 7.0 g, glycidyl methacrylate
5.4 g, β-methacryloyloxyethyl hydrogen hydrogen succinate 1.05 g, glycerol dimethacrylate (N
0.8 g of K ester 701: described above) and 2,2'-azobis- (2,4-dimethoxyvaleronitrile) (V-65:
A monomer solution consisting of 0.02 g was prepared. Nitrogen gas was introduced into this solution to remove dissolved oxygen. Porous ceramics (Bridgestone ceramic foam #
10 ml of the above-prepared monomer solution was impregnated into 10 ml of 40 average pore diameter (3000 μm), and the excess monomer solution was blown off with nitrogen gas to remove. The ceramic foam piece was placed in a glass container, heated to 60 ° C. in a nitrogen atmosphere, and subjected to a polymerization reaction under a nitrogen atmosphere for 2 hours. 50% heated to 50 ° C to remove residual monomer and 1-hexanol after completion of polymerization reaction
It was washed with an aqueous methanol solution and finally with distilled water.

Further, after replacing the ceramics with dioxane, 1.5 ml of a dioxane solution containing 1.5 g of a diethylamine solution was added dropwise thereto, and amination was performed at 75 ° C. for 4 hours. After the reaction,
The ceramic foam pieces were sufficiently washed with a 50% aqueous methanol solution and then washed with distilled water. The amount of coated polymer was 8.7%.

Estimated amount of positively chargeable functional groups is about 1.45 meq
/ g, the amount of negatively chargeable functional groups is about 0.19 meq / g
It is. The surface area was 90 cm ² / ml and the porosity was about 87%. The corresponding positively charged monomer unit is diethylamino-2-hydroxypropyl methacrylate,
The CLOGP value is 1.116 and the corresponding negatively charged monomer unit is β-methacryloyloxyethyl hydrogen succinate, which has a CLOGP value of 0.866.

(Comparative Example-1) The spherical cell culture carrier of Comparative Example was produced by the method described below. A thermometer, a cooling tube, a nitrogen introducing tube, and a stirring blade were attached to a 300 ml four-necked flask, and 30 g of calcium chloride / dihydrate, 50 ml of a 3% aqueous solution of polyvinyl alcohol, and 85 ml of ion-exchanged water were put therein and stirred. The polymerization reaction was performed under a nitrogen atmosphere. Into this, 60 g of 1-hexanol, 30 g of cyclohexanol, 13.2 g of polyethylene glycol methacrylate (PE-350: described above), 6.0 g of glycidyl methacrylate, 0.8 g of polyethylene glycol dimethacrylate (4G: described above), 2,2 as a polymerization initiator An organic solvent in which 0.02 g of '-azobis- (2,4-dimethylvaleronitrile) (V-65: described above) was dissolved was added into the flask.
The temperature was gradually raised, and a polymerization reaction was carried out at 65 ° C. for 5 hours. After completion of the reaction, the polymer was placed on a Buchner funnel and washed with a 50% aqueous methanol solution. The polymer was put back into the flask. This operation was repeated and washed four times until the odor of 1-hexanol and cyclohexanol disappeared. The obtained polymer was a colorless transparent sphere. The polymerization yield is
87%. Using a standard sieve, this polymer is
The particle size was adjusted to 130130 μm. The following reaction was further performed using this polymer. The polymer was thoroughly washed with 1,4-dioxane and replaced in 30 ml (in water). This was put in a 300 ml flask, and 30 ml of dioxane was further added. 15 ml of a dioxane solution containing 5.0 g of ethanolamine was added dropwise thereto, and an amination reaction was performed at 70 ° C. for 4 hours. After the reaction,
The polymer was thoroughly washed. Elemental analysis and titration revealed that the amine had a functional group content of 1.46 meq / g. The swelling degree of the polymer in a PBS solution is 17.9 ml / g, and the average particle size is 18
4 μm, specific gravity was 1.03 g / ml.

The corresponding positively charged monomer unit is hydroxyethylamino-2-hydroxyethyl methacrylate, which has a CLOGP value of -0.669.

(Cell culture experiment) A cell culture experiment was performed using each of the cell culture carriers manufactured in Examples 1, 2, 3, and 6 and Comparative Example-1. Each of the above-mentioned cell culture carriers was sufficiently washed with distilled water, washed with a PBS solution, and further replaced twice with Dulbecco's Eagle's denaturing medium.

For cell culture, the cells were cultured in an e-RDF medium (manufactured by Far East Pharmaceutical Co., Ltd.) containing 10% fetal bovine serum (manufactured by Mitsubishi Chemical Corporation). The cells subjected to the cell culture are CHO-K1 (Chinese hamster uterine ovary) cells, Vero (African green monkey kidney) cells, and MDCK (Coca spaniel kidney) cells. Microcarrier culture was performed as follows. The microcarriers produced in Examples-1, 2, 3, 6 and Comparative Example-1 were replaced with Dulbecco's Eagle modified medium.
This microcarrier 15 is placed in one spinner flask.
Then, the mixture was added and sterilized in an autoclave at 121 ° C. for 30 minutes. 20 ml of fetal calf serum and eRDF medium were added thereto to make 295 ml. Furthermore, the above CHO-K1 cells, Vero cells, MDCK cells
2.5 × 10 ⁷ cells / ml were added, and the mixture was continuously stirred at 25 rpm to start culture. The medium was changed with 5% serum from day 4 and 2 days from day 6.
The medium was replaced with% serum, and serum-free culture was started on the eighth day. In this state, serum-free culture was performed for 100 days. The results are shown in Table-4.

In addition, the result in a table | surface showed the evaluation result by the following reference | standard.

++: Very good ++: Good +: Cell adhesion and culture are observed, but not very good.

FIG. 1 shows a photograph of Vero cells cultured for 84 days using the carrier of Example 1. The cells have good extensibility even when cultured for a long period of time.

FIG. 2 shows that CHO-K1 cells were prepared using the carrier of Example-1.
The photograph at the time of culturing for a day is shown. Even when cultured for a long time
The extensibility of CHO-K1 cells is good.

FIG. 3 shows a photograph of Vero cells cultured for 69 days using the carrier of Comparative Example 1. Many detached and aggregated cells are observed on the microcarrier.

(Effects of the Invention) According to the present invention, since at least a positively-charged chemical residue and a negatively-chargeable chemical residue coexist on the surface of a support, cell attachment and proliferation are good. Not only that, but also in particular, it has been found that cells are hardly detached even after long-term culture.

In addition, the polymerizable monomer that constitutes the culture carrier is a transparent liquid, so it can be impregnated and covered with structures of various shapes, and it is optically transparent so that cells can be easily observed. Is mentioned. Further, since the polymer is a hydrophilic polymer, nonspecific adsorption of protein is hardly observed.

This makes it possible to produce carriers suitable for various uses, and enables efficient production of animal cell cultures and bioactive proteins, monoclonal antibodies, vaccines, viruses, and the like secreted thereby. .

BRIEF DESCRIPTION OF THE DRAWINGS FIGS. 1 and 2 show micrographs (magnification × 100) of the morphology of Vero cells and CHO-K1 cells cultured for 84 days using the carrier of Example 1, respectively. FIG. FIG. 3 is a drawing showing a micrograph (photograph magnification × 100) of Vero cells cultured for 69 days using the carrier of Comparative Example 1.

──────────────────────────────────────────────────続 き Continuation of front page (56) References JP-A-2-291260 (JP, A) JP-A-64-10979 (JP, A) JP-A-63-226283 (JP, A) JP-A-63-226 JP-A-61-173580 (JP, A) JP-A-61-56073 (JP, A) JP-A-2-502244 (JP, A) JP-A-2-504221 (JP, A) (58) Field surveyed (Int.Cl. ⁶ , DB name) C12N 5/06

Claims (3)

(57) [Claims] 1. The support itself is made of a water-insoluble polymer having a polymerizable monomer as a constitutional unit, or the support surface is coated with the water-insoluble polymer, and the polymer is positively charged. A cell culture carrier comprising a chemical residue capable of being charged and a chemical residue capable of being negatively charged. 2. The cell culture carrier according to claim 1, wherein the polymerizable monomer is (meth) acrylic acid ester and / or (meth) acrylamide. 3. A CLOGP value of a monomer unit corresponding to a positively chargeable chemical residue is -1.5 to +2.0, the content is 0.5 to 3.0 meq / g of a water-insoluble polymer, and CLOGP of monomer units corresponding to negatively chargeable chemical residues
The value is -1.5 to +2.1, and the content is 0.01 to 2.0 meq / g of the water-insoluble polymer.
The carrier for cell culture according to claim.