JPH06153905A - Cell culture base and method for cell culture - Google Patents

Abstract

PURPOSE:To obtain a cell culture base which forms a cell aggregate not to cause dedifferentiation for a long period of time, is capable of eliminating floating cell aggregate, prevents necrosis of internal cells caused by aggregation of mutual floating cell aggregates, is capable of making cells cultured in vitro to develop functions of cells in vivo, maintaining the functions for a long period of time, is readily and inexpensively prepared and has high stability and to culture cells. CONSTITUTION:In a method of cell culture, a cell culture base having the surface of the base composed of a vinyl-based copolymer containing 10-90mol% 2-hydroxyethyl methacrylate is used, cell aggregate is formed and cell aggregate is bonded to the surface of the base and maintained to give a cell culture base. Cells are cultured by using the cell culture base.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a cell culture method and a cell culture substrate.

[0002]

2. Description of the Related Art With the rapid development of biotechnology fields such as artificial organs, it is an urgent requirement to express the function of cells in vivo in cells cultured in vitro and maintain it for a long period of time. Conventionally well-known cell culture substrates and cell culture methods include a monolayer culture method in which a cell culture substrate having a surface made of an adhesive protein such as collagen is used to form a monolayer with proliferation.

Further, in recent years, under the concept of attempting to express the function of cells by reconstructing tissues in vitro, a substrate for cell adhesion is provided in a culture container made of plastic which does not have negative charge. And a method for culturing hepatocytes in a spherical shape, which comprises culturing in a serum-free medium without adding an extracellular stromal substance to form spherical aggregates of floating cells that are aggregated (Japanese Patent Application Laid-Open No. HEI-1) No. 277486) or poly-N-p-vinylbenzyl-D-lactonamide, and then coated with this aqueous solution and culturing hepatocytes using a serum-free culture medium to obtain high functional expression. And a method for forming and culturing cell aggregates having long-term viability (Kazubayashi Kobayashi et al., Artificial Organ Vol. 19, pp. 1156-1160,
Three-dimensional culture methods such as 1990) have been proposed.

[0004]

However, in the monolayer culture method on the surface composed of the adhesive protein, it is difficult to maintain the cell function possessed in the living body, and the cells survive and proliferate. However, it rapidly dedifferentiates and loses its function. Further, the adhesive protein is expensive and has a problem in its stability.

In the culturing method of Japanese Patent Application Laid-Open No. 1-277486, suspended cell aggregates have the potential to maintain the function of cells in vivo for a long period of time, even though the suspended cell aggregates have a possibility of being suspended. There was a problem that the clumps aggregated into a large clump and the inner cells were necrotic within a short period of time. Also in the method of culturing hepatocytes on the surface coated with an aqueous poly-N-p-vinylbenzyl-D-lactonamide solution, the cell aggregates are easily separated from the surface of the cell culture substrate, and the same problems as described above are encountered. Have There is also a problem that the synthesis of poly-Np-vinylbenzyl-D-lactone amide is very complicated.

[0006]

Means for Solving the Problems The present inventor has formed a cell aggregate that does not undergo dedifferentiation for a long period of time, eliminates necrosis of internal cells due to aggregation of floating cell aggregates, and has the function of cells in vitro. A cell culture substrate that is easy to make, inexpensive, and highly stable, and that can be expressed in cells cultured in E. coli and maintained for a long period of time; By controlling the adhesive force of the vinyl copolymer by using an inexpensive vinyl copolymer and changing the surface condition, only the vinyl copolymer surface with a specific composition forms cell aggregates and the cell aggregates are formed. It was found that there was no peeling of the above, and the present invention was reached.

That is, according to the present invention, in the three-dimensional culture method, the surface of the substrate is composed of a vinyl-based copolymer (A) obtained by copolymerizing a polymerizable monomer (a) represented by the following general formula (1). An animal cell culture substrate (I), characterized by forming a cell aggregate and maintaining the cell aggregate adherent to the surface; and a cell aggregate on the animal cell culture substrate surface (I) An animal cell culture method characterized by culturing while maintaining adhesion. (However, R ₁ is a hydrogen atom or a methyl group, R ₂ is a carbon atom 1
4 straight or branched alkylene groups)

The three-dimensional culturing method of the present invention is not a method which is frequently used in a packed bioreactor, in which cells are held inside a porous substrate and cultivated in a three-dimensional high density. It is a method of culturing by forming cell aggregates adhered to the. Hereinafter, the cell culture substrate of the present invention will be described in detail. As the polymerizable monomer (a), 2-hydroxymethyl methacrylate, 2-hydroxyethyl methacrylate, 2-hydroxyethyl acrylate,
2-hydroxy isopropyl methacrylate etc. are mentioned. Particularly preferred are 2-hydroxymethyl methacrylate and 2-hydroxyethyl methacrylate.

The vinyl copolymer (A) obtained by copolymerizing the polymerizable monomer (a) is not particularly limited, but for example, methyl methacrylate, ethyl methacrylate,
Vinyl-based copolymer (A) obtained by copolymerizing with monomers such as dimethylaminoethyl methacrylate, diethylaminoethyl methacrylate, acrylic acid, methyl acrylate, methacrylic acid, methyl methacrylate and styrene.
Etc. It may be a copolymer with one kind of the above-mentioned monomer, or may be a vinyl-based copolymer (A) obtained by copolymerizing with one or more kinds of monomers. Further, either random or block copolymerization may be used.

To form a cell aggregate and to maintain the adhesion of the cell aggregate by adhering and maintaining the cell aggregate, the function of the cell in the living body can be expressed in the cell cultured in vitro and the culture can be performed for a long period of time. , The content of the polymerizable monomer (a) with respect to the vinyl-based copolymer (A) is 10 to 90 mol%
Must be within the range. When the content of the polymerizable monomer (a) is less than 10 mol%, the cells adhere to the surface of the copolymer to form a monolayer, and form a cell aggregate without forming a flat surface completely different from that in the living body. It becomes a cell morphology. In this flat cell morphology, although it survives and proliferates, it is rapidly dedifferentiated, and the function originally possessed by cells in vivo cannot be expressed and maintained in vitro for a long period of time. When the content of the polymerizable monomer (a) is higher than 90 mol% based on the vinyl copolymer (A), cell aggregates are formed, but the cell aggregates adhere to the vinyl copolymer surface. It cannot be maintained and floats, cell aggregates aggregate to form a large aggregate, and the cells die in a short period due to necrosis of the inner cells.

The vinyl copolymer (A) can be polymerized by solution polymerization in the presence of an organic diluent and a polymerization initiator. The organic diluent is not particularly limited as long as it is inactive to a functional group such as a hydroxyl group, but toluene or the like is usually used. The amount of the organic diluent added to the monomer is preferably 0.5 to 20 times by weight the total amount of the monomer components. Further, as the polymerization initiator,
Benzoyl peroxide, lauroyl peroxide, azobisisobutyronitrile, azobisisovaleronitrile and the like are used, and the amount thereof is usually 0.01
Is in the range of 5.0% by weight. Preferably 0.01-
It is selected from the concentration range of 1.0% by weight.

The cell culture substrate (I) in the present invention is obtained by coating the vinyl-based copolymer (A) on various shaped materials or by coating the vinyl-based copolymer (A) on various materials. It can be created by molding. Examples of the material to be coated include synthetic polymer materials, natural polymer materials and inorganic materials.

Examples of the synthetic polymer material include olefin polymers such as polyethylene, polypropylene, chlorinated polyethylene and ionomer, fluorine resins such as polytetrafluoroethylene and polyvinylidene fluoride,
Styrene resins such as polystyrene, acrylic resins such as polymethylmethacrylate, polyvinyl alcohol, polyvinyl acetate, polyvinyl acetal, polyacrylonitrile, polyvinyl chloride, polyvinylidene chloride,
Examples include polyester resins such as polycarbonate, polyacrylate, polyphenylene oxide, polyethylene terephthalate and polybutylene terephthalate, various polymers or copolymers such as epoxy resin, polyamide, polyimide, polysulfone, silicone resin and polyurethane, or blends thereof.

Examples of natural polymer materials include cellulose, cellulose derivatives, chitosan and chitin.

Examples of inorganic materials include glass and ceramics.

Examples of the shape of the material to be coated or the shape to be molded include petri dishes, flasks, films, tubes, fibers, fine particles, non-woven fabrics, porous foams, porous beads, hollow fibers and the like. Among these shapes, porous materials such as hollow fibers and porous beads that can be easily applied to high-density mass-culturing devices are used in order to maintain high cell function and perform high-density mass-culturing. preferable. In the case of a small scale, the shape of a petri dish, a bottle, etc. is preferable from the viewpoint of easy handling. In the case of coating, the film thickness is not particularly limited as long as the surface characteristics are changed to the properties of the copolymer and the coating has durability.

Next, the cell culture method of the present invention will be described in detail. The cell culture method of the present invention can be performed by seeding cells on the cell culture substrate of the present invention. After the cell seeding, the cells form a three-dimensionally densely aggregated cell aggregate, and adhere and maintain the cell culture substrate surface.

The cells to be cultured include, among animal cells, cells of a primary culture system in which it is difficult to highly express and maintain the functions of the cells in vivo in vitro, and cells in vivo that are not difficult to culture. The cell line and the like in which it is desired to highly express the function originally possessed by the cell in vitro are mentioned.

Examples of the cells of the primary cell line include hepatocytes, mammary epithelial cells, pancreatic islet cells, osteoblasts, osteoclasts, chondrocytes, renal tubular epithelial cells, glomerular cells, vascular endothelial cells, Langerhans cells. , Various types of fibroblasts such as bile duct epithelial cells and skin fibroblasts. These may be cancerous cells. Examples of the established cell lines include cell lines derived from liver cancer (HepG2, Huh-).
3), cell lines derived from various cancer cells such as cell lines derived from pancreatic cancer, fibroblast cell lines derived from normal skin, cell lines derived from various normal cells such as normal vascular endothelial cell lines derived from normal vascular endothelium Can be exemplified. These cells may be cultivated by one kind, but one or more kinds of cells may be cultivated at the same time. The mixing ratio in this case can be appropriately changed,
The combination is not limited to cells of the primary cell line, and cell lines can be freely selected and used.

As the culture solution, various culture solutions are used depending on the type of cells to be cultured. Specifically, insulin (10 μg / m 2) was added to Dulbecco's modified Eagle medium.
l), epidermal growth factor (10 ng / ml), and ascorbic acid (20 μg / ml) are added to adjust the hydrogen ion concentration to 10 ⁻⁷ mol / l. In addition, a method of using the culture medium may be exemplified, in which after the culture is started in the culture medium to which the serum is not added, the culture medium is switched to the culture medium to which the serum is added to perform the culture.

The culture environment is a temperature suitable for maintaining the function of cells,
Culture can be performed under conditions such as carbon dioxide concentration. For example, culturing in an atmosphere having a temperature of 37 ° C., a humidity of 100% and a carbon dioxide gas concentration of 5% can be exemplified.

The cell culture method and the cell culture substrate can be used, for example, in animal experiments because the functions of cells in vivo can be expressed in cells cultured in vitro and maintained for a long period of time. As a substitute for cells, cells for the purpose of usage such as judgment of toxicity of chemical substances, search for useful substances, elucidation of cell function, culturing cells obtained from pathological tissues, screening effects of various drugs in vitro and utilizing for treatment It can be used for culture kits, mass production of useful substances produced by cells, construction of hybrid artificial organs, etc.

[0023]

EXAMPLES The present invention will be specifically described below with reference to Examples.
The invention is not limited to these examples.

Example 1 of base material preparation 10.4 parts of 2-hydroxyethyl methacrylate, 3.1 parts of dimethylaminoethyl methacrylate, 121.5 parts of toluene and 0.07 parts of azobisisobutyronitrile were charged and the temperature was 65 ° C. Polymerization was carried out for 8 hours. The precipitated copolymer was washed with toluene and then dried. As a result of NMR analysis, 80% of 2-hydroxyethyl methacrylate was obtained.
A vinyl-based copolymer containing mol% was obtained.

Next, the vinyl-based copolymer was dissolved in dimethylformamide at 2.0% by weight, and a spin coater (spin coater 1H-DX was used on a circular glass plate having a diameter of 50 mm).
Spin coating (2000rp) by Mikasa Co., Ltd.
m, 80 seconds) to obtain a cell culture substrate 1.

Substrate preparation example 2 Substrate preparation example except that 2.3 parts of 2-hydroxymethyl methacrylate, 8.0 parts of methyl methacrylate and 92.7 parts of toluene and 0.05 parts of azobisisobutyronitrile were charged. The same procedure as in 1 was performed. As a result of analysis by NMR, 20 mol% of 2-hydroxymethyl methacrylate was obtained.
The contained vinyl-based copolymer was obtained. Next, base material preparation example 1
Spin coating was performed in the same manner as above to obtain a cell culture substrate 2.

Comparative Substrate Preparation Example 1 The same procedure as in Substrate Preparation Example 1 was repeated except that 10 parts of 2-hydroxyethyl methacrylate, 90 parts of toluene and 0.05 part of azobisisobutyronitrile were charged. The obtained polyhydroxyethyl methacrylate was spin-coated in the same manner as in the substrate preparation example 1 to obtain a cell culture substrate 3.

Comparative Base Material Preparation Example 2 Polymerization was carried out in the same manner as in Base Material Preparation Example 1 except that 10 parts of methyl methacrylate was charged with 90 parts of toluene and 0.05 part of azobisisobutyronitrile, and then re-used with methanol. It was purified by precipitation. The obtained polymethylmethacrylate was spin-coated in the same manner as in Substrate preparation example 1 to obtain a cell culture substrate 4.

Comparative Base Material Preparation Example 3 Methyl methacrylate (10 parts), styrene (10 parts), toluene (100 parts) and azobisisobutyronitrile (0.1 parts) were charged, polymerization was carried out in the same manner as in the base material preparation example 1, and then methanol was prepared. Was purified by reprecipitation, and a copolymer of methyl methacrylate and styrene was obtained. Then, spin coating was performed in the same manner as in Example 1 for preparing a substrate to obtain a cell culture substrate 5.

Culture Example 1 After sterilizing the cell culture substrate 1, it was aseptically transferred to a polystyrene dish having a diameter of 60 mm and subjected to culture. The evaluation was performed using a primary culture system of rat liver parenchymal cells. Liver parenchymal cells were obtained from Wistar rats by a conventional in situ collagenase perfusion method (primary culture hepatocyte test method, 6-).
Page 10, Toshikazu Nakamura, Academic Publishing Center). The liver parenchymal cells used had a survival rate of 98% or more. Culture solution is 10%
Serum-containing medium was used (basal medium was Dulbecco's modified Eagle medium: insulin 10 μg / ml, epidermal growth factor 10 ng / ml, ascorbic acid 20 μg / ml)
ml, proline 20 μg / ml, dexamethasone 10 ^-6
M addition).

Liver parenchymal cells were 5 × 10 5 per 1 ml of the culture medium.
After adjusting the cell density to ⁵ cells, 4 ml of the cell suspension was seeded on a petri dish to which the cell culture substrate 1 was transferred, and hepatocyte culture was started. Twenty-four hours after the start of culture, the culture was switched to a serum-free culture medium and continued to culture. The culture solution was completely replaced by 4 ml every 24 hours.

For the evaluation, the amount of albumin production, which is a liver-specific function, was quantified daily by ELISA, and 1x liver parenchymal cells were evaluated.
The viability of hepatocytes was evaluated by the amount of albumin produced by 10 ⁶ cells in 24 hours. The cell aggregate formation rate [(the number of cells involved in cell aggregate formation / seeded cells) x 100] was evaluated 3 days after the start of culture, and the cell aggregate adhesion retention rate [on the 3rd and 20th days of culture] ((Number of formed cell aggregates-total number of detached cell aggregates) / number of formed cell aggregates) x 100] was evaluated.

Culture Example 2 The same method as in Culture Example 1 was used except that the cell culture substrate 2 was used.

Comparative Culture Example 1 The procedure of Culture Example 1 was repeated except that the cell culture substrate 3 was used.

Comparative Culture Example 2 The procedure of Culture Example 1 was repeated except that the cell culture substrate 4 was used.

Comparative Culture Example 3 The procedure of Culture Example 1 was repeated except that the cell culture substrate 5 was used.

Comparative Culture Example 4 The same method as in Culture Example 1 was used except that a commercially available primary adish 3802 (60 mm in diameter, manufactured by Becton & Dickington) having no negative charge was used as the cell culture substrate. .

The above-mentioned culture examples 1 and 2 and comparative culture examples 1-
The evaluation results of No. 4 are shown in Tables 1 and 2 below.

[0039]

[Table 1]

(1): Ability to develop maximum function → number of seeded hepatocytes 1
Converted to X10 ⁶ cells per day during the culture period
Amount of albumin produced on the day when the maximum amount of albumin was produced (μg) (2): Maintenance period of function expression ability → converted to per 1 × 10 ⁶ cells, and the number of days of culture in which albumin production of 10 μg or more per day continues ( Day)

In the culture method using the cell culture substrate of the present invention, the maximum function expressing ability is about 1.5 times higher than that of the comparative example.
The function maintenance period has also been significantly extended.

[0042]

[Table 2]

(1): Cell aggregate formation rate = (number of cells involved in cell aggregate formation / seeded cells) × 100 (2): Cell aggregate adhesion retention rate = ((number of formed cell aggregates− Total number of detached cell aggregates) / number of formed cell aggregates) x 100

In the culture method using the cell culture substrate of the present invention, almost 100% of cell aggregates are formed in the seeded cells, and about 80% of the cells are formed even after 20 days of culture. Adhesion is maintained on the surface of the culture substrate. On the other hand, although cell aggregates are formed on the cell culture substrate 3, only about 10% of the cell aggregates are adhered and maintained on the surface of the cell culture substrate on day 3 of culture. In other comparative cell culture substrates,
Cell aggregates could not be formed and cells adhered to the monolayer.

[0045]

INDUSTRIAL APPLICABILITY The present hepatocyte culturing substrate and cell culturing method allow the function of cells in vivo to be expressed in cells cultured in vitro and maintained for a long period of time. , As an alternative to animal experiments, to determine the toxicity of chemical substances, search for useful substances, elucidate cell functions, cultivate cells obtained from pathological tissues, screen the effects of various drugs in vitro, and use them for therapeutic purposes. The cell culture kit described above can be used for mass production of useful substances produced by cells, construction of hybrid artificial organs, and the like.

Claims (3)

[Claims] 1. A three-dimensional culture method, wherein the substrate surface is
An animal cell culture substrate (I) comprising a vinyl copolymer (A) obtained by copolymerizing a polymerizable monomer (a) represented by the following general formula (1). (However, R ₁ is a hydrogen atom or a methyl group, R ₂ is a carbon atom 1
4 straight or branched alkylene groups. ) 2. The animal cell culture medium according to claim 1, wherein the content of the polymerizable monomer (a) with respect to the vinyl copolymer (A) is in the range of 10 to 90 mol%. Material (I). 3. A method for culturing an animal cell, wherein by culturing cells on the surface of the animal cell culture substrate (I) according to claim 1, cell aggregates are formed to form the animal cell culture substrate (I). A method for culturing an animal cell, which comprises culturing while keeping the cell aggregate adhered to the surface.