JPH0833474A - Culture base for adhered animal cell - Google Patents

Abstract

PURPOSE:To obtain a culture base for adhered animal cells, composed of an inexpensive, physically stable and high heat-resistant material and which can adhere, extend and proliferate the animal cells in high density and with very high efficiency and thereby can produce a desired metabolite product efficiently therefrom. CONSTITUTION:This culture base for adhered animal cells comprises a fixing bed as a fiber accumulating body and a culture bed formed on the fiber surface wherein the fixing bed is composed of fibers resisting to heat above 120 deg.C and having 20-60mum filament diameter. These fibers entangle with each other to form the fiber accumulating body so that the fiber surface totals over 10-30m<2>/m<3> and the void volume is higher than 90%, while the culture bed contains a layer of an acidic synthetic polymer.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a culture substrate for adherent animal cells, and more particularly to a culture substrate capable of high-density culture of adherent animal cells.

[0002]

2. Description of the Related Art Conventionally, it has been practiced to cultivate various animal cells in a container, obtain a physiologically active substance produced by the animal cells in the process of proliferation and put them into practical use as a medicine. The production of physiologically active substances by culturing animal cells has recently attracted particular attention. In such production, it is important to produce the physiologically active substance selectively, continuously, and efficiently, and many culture techniques have been developed up to now.

A large number of animal cell culture techniques are roughly classified into "floating growth method" in which animal cells are grown in a state of being suspended in an aqueous solution of a medium, and animal cells are polymerized with acrylates or dextran, A "microcapsule growth method" in which a microcapsule is coated with a porous film made of a film-forming substance such as cellulose or chitin and embedded into microcapsules, and the microcapsules are grown in a state of being suspended in an aqueous solution of a medium. "Microbeads growth method", in which microbeads made of ceramic or the like and having a diameter of about 200 μm are adsorbed and fixed, and then grown in suspension. There is an "adhesion-carrying growth method" in which an aqueous solution is supplied to grow animal cells.

Of these, as a culture method belonging to the "adhesion-carrying and multiplying method", for example, Japanese Patent Application Laid-Open No. 4-63584 discloses an invention called "bioreactor apparatus". According to this bioreactor apparatus, It is possible to produce a physiologically active substance selectively, continuously and efficiently. In order to achieve the object, in the present invention, the device structure is devised and the culture substrate (carrier) placed in the device to attach the animal cells is also devised. That is, in the invention, a layer of a culture bed showing high affinity and adhesiveness to animal cells such as atelocollagen, polyvinyl alcohol having a photocrosslinking reactive group, and a polypeptide in a reticulated fiber such as a non-woven fabric is provided in the invention. It is provided as a culture substrate for animal cells.
In addition, examples of using a nonwoven fabric as a substrate for culturing animal cells include WO88 / 02398 and JP-A-64-34.
There are various disclosures in Japanese Laid-Open Patent Publication No. 276 and Japanese Laid-Open Patent Publication No. 2-154685, but in these techniques, the nonwoven fabric is a fiber having a thickness of 1.0 d (corresponding to about 0.6 μm) or less. It is extremely thin, and the fiber diameter, porosity, and thickness that make up the non-woven fabric are unsuitable for cell attachment and culture, and the non-woven fabric material and fiber surface have inadequate polarity, hydrophilicity, and smoothness. For this reason, the coating of the culture bed for adhering and culturing cells on the fiber surface becomes non-uniform, and the purpose for high-density culturing of cells cannot be sufficiently achieved.

Further, as a conventional animal cell culture bed,
In general, collagen, gelatin, fibronectin, laminin, lectin, fibrin or fibrinogen was applied to the fixed bed after applying a natural polypeptide-based polymer, was formed by insolubilization treatment,
The culture bed using these natural polypeptide-based polymers is
Not only was it not suitable for autoclave sterilization because it was poor in physicochemical stability, was susceptible to metamorphosis, and had poor heat resistance, it was extremely expensive. Therefore, it has been required to supply an excellent animal cell culture bed that is industrially inexpensive.

[0006]

DISCLOSURE OF THE INVENTION As a result of intensive studies to solve the above-mentioned problems of the prior art, the present inventors have developed a substrate capable of culturing animal cells at high density as described below. The present invention has been successfully achieved, and has achieved a highly efficient biochemical reaction that is several times to several tens of times higher than that of conventional static culture or culture using a porous carrier, which has led to the present invention. It is a thing. That is, the present invention comprises a fixed bed which is a fiber assembly and a culture bed of adherent animal cells provided on the fiber surface of the fixed bed, the fixed bed having a heat resistance of 120 ° C.
A substrate for culturing adherent animal cells, which is a fiber assembly composed of fibers having a fiber diameter of 20 to 60 μm and having a fiber surface area of 10 to 30 m ² / m ³ and a porosity of 90% or more. In the above, the culture bed is a culture substrate for adherent animal cells, which is a layer containing an acidic synthetic polymer having an acidic group such as a sulfonic acid group or a carboxyl group.

Hereinafter, the present invention will be described in detail. The culture substrate for adherent animal cells of the present invention basically comprises a fixed bed made of a fiber aggregate and a culture bed coated on the surface of each fiber. Various animal cells to be cultured have a size of several microns, and these animal cells adhere to the culture bed,
A fiber diameter suitable for fixation and extension is about 20 to 60 μm, and is composed of monofilaments having a smooth surface. The fiber packing density is 10% or less, the thickness is 10 mm or less, and the fiber surface area is 10.
A fiber assembly having a physical property of ˜30 m ² / m ³ (for example, such a nonwoven fabric) is used as a fixed bed of adherent animal cells. The material of this fiber assembly has the above-mentioned physical properties, has heat resistance such that it can be sterilized by autoclave, has compatibility with the culture bed to be coated on the surface, and can be applied uniformly. As synthetic polymers, polyester, polyamide,
Polyurethane, polyvinyl alcohol, polyacrylonitrile, polytetrafluoroethylene, polystyrene,
Rayon, methyl methacrylate, polysulfone, polyphenylene sulfide, polyphenylene sulfoxide,
Materials such as polycarbonate, polyphosphazene, polyacetal, polyvinyl chloride, and hydrophilic polyolefin are treated with plasma or synthetic fiber-based non-woven fabric to which anionic hydrophilicity is imparted. Further, as the natural polymer, a non-woven fabric made of collagen, chitosan, chitin, or cellulosic fibers that has been insolubilized to have heat resistance is also used. However, considering the characteristics and the price, a non-woven fabric of polyester synthetic polymer or polyamide synthetic polymer is preferable.

All the surfaces of the fibers of these fibrous aggregates are
As a culture bed, a thin layer of an acidic synthetic polymer having an acidic group such as a sulfonic acid group or a carboxyl group is applied and then insolubilized. The acidic polymer having a sulfonic acid group or a carboxyl group is a polymer electrolyte, and examples of the polymer electrolyte having a sulfonic acid group include polyvinyl sulfonic acid, polystyrene sulfonic acid, and polyacrylic acid having a sulfonic acid group in a side chain. There are derivatives and polyacrylic acid amide derivatives. These polymer electrolytes are soluble in water or an aqueous alcohol solution and have a molecular weight of tens of thousands to 1,000,000. Further, as the polymer electrolyte having a carboxyl group, an aliphatic carboxylic acid polymer such as polyacrylic acid or polymethacrylic acid or a copolymer thereof,
There are aromatic carboxylic acid polymers such as polyacrylic acid amide derivatives having a carboxyl group and polystyrene derivatives. These carboxyl group-containing polyelectrolytes are soluble in organic solvents, and their molecular weights are still in the range of tens of thousands to 1
It is about, 000,000.

In order to form a thin layer of these polymers as a culture bed on the fiber surface of the fiber assembly which is the fixed bed of the culture substrate, for example, in the case of the above-mentioned polymer having a sulfonic acid group, this sulfonic acid group is used. A 1: 1 (weight ratio) mixture of the containing polymer and polyvinyl alcohol was added to water or methanol,
A concentration of an aqueous alcohol solution of a lower aliphatic alcohol soluble in water such as ethanol or propanol is 0.
A solution of 1 to 10% by weight is used as a coating liquid, and usually, a fiber aggregate which is a fixed bed is immersed in the above coating liquid, and then the coated object is left under reduced pressure for about 1 hour to form air bubbles inside the fiber. Are completely removed so that the coating solution is applied to the entire surface of the fiber, so that a uniform thin layer of polymer is formed. After that, the excess coating liquid is squeezed out and removed, and heat treatment is performed at a temperature of 120 ° C. for 2 hours to partially dehydrate and condense between the sulfonic acid group and the hydroxyl group of polyvinyl alcohol to form a cross-linking bond between the two polymers, thereby making them insoluble. Let Then, it is immersed in a 5 wt% sodium chloride aqueous solution to convert the sulfonic acid group into a sodium salt, washed with water, and dried at 80 ° C. for 2 hours to obtain a culture bed. The thickness of the culture layer is preferably about several μm to several tens of μm.

On the other hand, in the case of the above-mentioned carboxyl group-containing polymer, the carboxyl group-containing polymer is dissolved in a solvent such as water, alcohol, acetone or various ketones at a concentration of 0.
A solution of 1 to 10% by weight is used as a coating liquid, and usually, a fiber aggregate which is a fixed bed is immersed in the above coating liquid, and then the coated object is left under reduced pressure for about 1 hour to form air bubbles inside the fiber. Are completely removed so that the coating solution is applied to the entire surface of the fiber, so that a uniform thin layer of polymer is formed. After that, the excess coating solution is squeezed out and removed, dried at a temperature of 80 ° C. for several hours, and then immersed in a 0.3% polyethyleneicine aqueous solution for about 30 minutes to be condensed and insolubilized to obtain a culture bed. The thickness of the culture layer is preferably about several μm to several tens of μm.

Animal cells to be cultured using the culture substrate of the present invention can be freely selected according to the target metabolite, and examples thereof include T cells, B cells, killer cells, human tumor cells and fibroblasts. Cells, lymphocytes, lymphoblasts, EB virus mutant cells, hepatocytes, kidney cells, epidermal cells, bone marrow cells, macrophages, vascular endothelial cells, smooth muscle cells, hepatocytes, pancreatic β cells, epithelial cells, small intestine cells, Examples include mammary gland cells, mammary gland epithelial cells, salivary gland cells, thyroid cells, living body-derived skeletal muscle cells, and human skin cells. These cells are mixed and dispersed in a solvent such as water at a concentration of about 10 ³ to 10 ⁵ cells / ml and attached to the culture bed of the culture substrate.

[0012] Examples of the culture solution supplied to the animal cells to be further cultured include various essential amino acids, various vitamins,
Examples include sugars such as glucose, serum components such as beef tallow serum medium, serum-free medium, and human serum medium. These are 1
It is preferably supplied in the form of an aqueous solution having a concentration of about 100 g / l. Furthermore, the substrate to be supplied to animal cells can be freely selected depending on the target metabolite, and examples thereof include various essential amino acids, various vitamins, sugars such as glucose, and serum components. ,
It is preferably supplied in the form of an aqueous solution having a concentration of about 1 to 100 g / l. The same substance may be used as the culture component and the substrate, but the culture component is for growing animal cells, while the substrate is for obtaining a metabolite according to the purpose from the animal cell. Is different.

Examples of metabolites that can be produced from the culture bed in this manner include therapeutic vaccines, insulin, renin, immunoglobulin M, fibrinogen, albumin, lymphokines, interferons,
Examples include physiologically active substances such as urokinase, growth hormone, monoclonal antibody, cancer antibody, hormone, cell growth factor, and various enzymes, which are extracted and purified from a metabolic product solution.

When a fiber assembly composed of monofilaments having a fiber diameter of 20 to 60 μm and having a smooth surface is used for a fixed bed, the curvature of the fiber surface is suitable for cell attachment, proliferation and spreading. Then, the cells are first adhered and immobilized on the left and right ends of the fiber, then gradually spread along the surface of the fiber to the central part along the fiber surface, and become wound around the fiber, and eventually the entire fiber grows. It will be covered with the cells that you made. Then, as the proliferation further progresses, the proliferation can be observed in a state where cells are stacked in multiple layers. The nonwoven fabric has a porosity of 90% or more and a fiber surface area of 10 to 30 m ²
If the thickness is less than 10 m / m ³ , the porous structure is suitable for uniform supply and diffusion of fresh medium and oxygen to the inside of the substrate and is just suitable for high-density culture of cells.

[0015]

When a thin bed of an acidic synthetic polyelectrolyte containing an acidic group characteristic of the present invention is used as a culture bed together with a fixed bed using such a porous fiber assembly, animal cells are used. Since it strongly adheres to and fixes it on the culture bed, there is little effect on cells due to the flow of a liquid such as a medium during culturing or inactivation of cells due to mutual contact of cells, and a fresh culture medium or oxygen is constantly produced by the reflux culture method. The desired final physiologically active substance can be obtained while continuously removing the cell growth inhibitor that is supplied to the cells and is produced, from the system. Although the culture substrate of the present invention has a desired strength and thermal stability and is made of an inexpensive synthetic polymer as a material, it is possible to culture adherent animal cells at a high density in this manner.

[0016]

EXAMPLES The method of the present invention will now be described specifically and in detail based on examples. However, the content of the present invention is not limited to these. Example (1) Preparation of culture substrate (sample) High-melting point polyester fiber having a fiber diameter of 55 μm (melting point 220)
° C.) and 90 wt% and the low-melting-point polyester fiber having a fiber diameter of 39μm (melting point 120 ° C.) made of a fiber aggregate containing 10% by weight, the fiber surface area porosity 90% or more 30 m ² /
The polyester non-woven fabric having a thickness of m ³ and a thickness of 6 mm was washed with distilled water (water temperature: 60 ° C.) until no leached substance was observed from the non-woven fabric, and then dried at a temperature of 80 ° C. for 6 hours.

On the other hand, a mixture of polystyrene sulfonic acid (sulfonic acid group content 4.5 meq / g, molecular weight about 100,000) and polyvinyl alcohol (average degree of polymerization: 1700, complete hydrolyzate) (weight ratio 1: 1). 1% by weight aqueous solution was prepared, and in this aqueous solution, the washed and dried non-woven fabric cut to a predetermined size was immersed under reduced pressure for about 1 hour to completely remove the air bubbles inside the non-woven fabric. The copolymer solution was uniformly applied to all surfaces. After that, take out the coated non-woven fabric, squeeze out the excess solution, and keep the temperature at 120 ° C.
The mixture was heat-treated for 2 hours in order to cause cross-linking between both polymers to make them insoluble. Then, after cooling to room temperature, it was immersed in a 5% by weight sodium chloride aqueous solution for 5 hours to convert it into a sodium salt of sulfonic acid, washed with water 5 times repeatedly, and dried at 80 ° C. for 2 hours. In this way, a culture substrate (Sample 1) was prepared in which a thin layer of a strongly acidic synthetic polymer having a sulfonic acid group was uniformly formed on the entire surface of the nonwoven fabric fiber.

Further, the washed and dried other part of the non-woven fabric is immersed in a 0.2% by weight aqueous solution of polyacrylic acid (average molecular weight: about 450,000) under reduced pressure for about 2 hours to form air bubbles inside the non-woven fabric. Was completely removed, and the polymer solution was uniformly applied to the entire surface of the fiber. Then, the coated nonwoven fabric was taken out, excess solution was squeezed out, and dried at a temperature of 80 ° C. for 2.5 hours. Then, it was immersed in a 0.5% polyethyleneimine aqueous solution at room temperature (25 ° C.) for 30 minutes to carry out an insolubilization treatment by a condensation reaction. The nonwoven fabric was taken out, excess solution was squeezed out, and heat treatment was performed at a temperature of 80 ° C. for 2.5 hours to complete the condensation reaction to uniformly form a thin layer of a synthetic polymer having a weak acidic group (Sample 2). )created.

(2) Sterilization of culture substrate Each sample of the culture substrate prepared as described above was washed 3 times with distilled water and 2 times with a phosphate buffer and ethanol, and then dried in a dryer (temperature: 60 ° C). And dried for 1 hour. Filling and setting each sample in a flat plate bioreactor,
Fill with phosphate buffer and autoclave (temperature 1
Sterilization was performed for 15 minutes at 21 ° C. and a pressure of 2 kg / cm ² . (3) Adhesion and culture of animal cells on culture substrate Next, the phosphate buffer was removed from the bioreactor as much as possible, and then 1% with DMEM medium containing 10% by weight of calf serum.
After pretreatment for 1 day and then removing this medium, a cell inoculation solution containing 3 × 10 ⁵ cells / ml of human renin-producing γ-CHO-Ar2 cells was introduced into the cells from the bottom of the bioreactor to culture the culture substrate. The cells were completely immersed in the inoculation solution and allowed to stand for 12 hours to allow the cells to adhere to the culture bed.

After the cells were attached, a 5% by weight aqueous solution (medium) of calf serum containing 1 g / l glucose was circulated from a medium tank prepared in advance at a flow rate of 11 cm / hour. During the circulation, cell culture was carried out for 188 hours while the medium was exchanged by adding 40 ml of fresh medium every 20 to 30 hours as the glucose concentration decreased due to cell culture and growth.

(3) Measurement after Cell Culture After the cell culture, the sugar consumption rate and the cell concentration in the culture bed were measured for each sample, and the following results were obtained. In the culture substrate of Sample 1 (culture bed: thin layer of polystyrenesulfonic acid-polyvinyl alcohol-based polymer), the cell concentration was 7.23 × 10 ⁵ cells / ml carrier, and the final sugar consumption rate was 2.55 × 10 ^−4. It was M / hour. Also, sample 2
In (culture bed: thin layer of polyacrylic acid type polymer), the cell concentration was 6.15 × 10 ⁵ cells / ml carrier, and the final sugar consumption rate was 2.32 × 10 ⁻⁴ M / hour. The culture beds of these culture substrates are all atherocollagen-based culture carrier membranes (the final sugar consumption rate is 2.
64 × 10 ⁻⁴ M / hour), and showed comparable or superior effects. From the above results, it was confirmed that the culture bed made of the synthetic polymer according to the present invention is suitable for cell attachment, spreading, and culture, and enables high-density cell culture.

[0022]

EFFECTS OF THE INVENTION According to the present invention, an acidic polymer substance having a strongly acidic sulfonic acid group and a weakly acidic carboxyl group is applied to the fiber surface of a fiber assembly as a fixed bed of a culture substrate to insolubilize it. The culture substrate thus obtained is inexpensive as a raw material, has stable physical properties, and has high heat resistance. Moreover, when culturing animal cells for experiments, it adheres and spreads animal cells with extremely high efficiency and proliferates at high density. Therefore, the useful effect that the desired metabolite can be efficiently produced is exhibited.

Claims (2)

[Claims] 1. A fixed bed, which is a fiber assembly, and a culture bed for adherent animal cells provided on the fiber surface of the fixed bed, the fixed bed having a heat resistance of 120 ° C. or higher and a fiber diameter of 20 to 6
The fiber has a surface area of 10 to 30.
In a culture substrate for adherent animal cells, which is a fiber aggregate formed by intertwining with each other with m ² / m ³ and a porosity of 90% or more, the culture bed is a layer containing an acidic synthetic polymer. And a culture substrate for adherent animal cells. 2. The adherent animal cell culture substrate according to claim 1, wherein the synthetic polymer contains at least one of a sulfonic acid group and a carboxyl group.