JP6369044B2 - Cultivation apparatus and manufacturing method thereof - Google Patents

Description

  The present invention relates to a culture apparatus and a method for producing the same.

  A wide variety of plastic containers are used to store, weigh, prepare, and fractionate biomaterials such as amino acids, peptides, proteins, nucleic acids, and cells (such as IPS cells and ES cells) and suspensions thereof. However, if the aforementioned biomaterial is brought into contact with a plastic container, a hydrophobic interaction may occur between the hydrophobic portion of the biomaterial and the plastic surface, and the biomaterial may migrate or be adsorbed to the container. . This becomes a big problem when the sample is very small for high sensitivity measurement. Furthermore, if the hydrophobic part of the biomaterial is stabilized at the interface on the plastic surface, the higher-order structure of the hydrophobic part may be deformed and the function originally possessed by the biomaterial may not be expressed.

For this reason, polypropylene (PP) having relatively low hydrophobicity is often used as the base material of the plastic container used for the above-described applications, rather than polystyrene (PS) having high hydrophobicity. However, since the hydrophobicity is not sufficiently low even with a PP base material to solve such a non-specific adsorption problem of the biomaterial, a hydrophilic polymer such as a polyvinyl alcohol (PVA) is further obtained. And a method of reducing the adsorption of biomaterials by coating with a surfactant or the like. (For example, Patent Document 1 and Patent Document 2) However, in these methods, the sample may be contaminated by the elution of the hydrophilic compound in the coding layer into the solvent contained in the medium containing the biomaterial. In addition, water-soluble polymers such as PVA are manufactured based on materials derived from fossil resources, and there is also a problem that an environmental load cannot be avoided.

  On the other hand, in recent years, with the aim of solving the problem of fossil resource depletion, development of functional materials using biomass, which is an environmentally friendly material that can be used continuously, has been actively conducted. Among them, cellulose, which is the main component of wood, is a natural polymer material accumulated in large quantities on the earth, and is expected as a key material for shifting to a resource recycling society. In wood, dozens or more of cellulose molecules are bundled to form fine fibers (microfibrils) having a high crystallinity and a nanometer order fiber diameter, and many fine fibers are hydrogen-bonded to each other. Cellulosic fibers are formed to become a plant support. Since it has such a stable structure, natural cellulose fibers contained in wood are insoluble except for special solvents, have poor moldability, and are difficult to handle as functional materials. Therefore, attempts have been actively made to use cellulose fibers in wood by making them fine (nanofibers) until the fiber diameter reaches the nanometer order.

As one of the techniques for converting cellulose fibers in wood into nanofibers, cellulose fibers are converted to 2,2,6,6-tetramethylpiperidinyl-1-oxy radical (TEMPO), which is a relatively stable N-oxyl compound. ) Has been reported as a catalyst (TEMPO oxidation) (for example, Patent Document 3). The TEMPO oxidation reaction can be chemically modified in an environmentally friendly manner that proceeds in water, normal temperature, and normal pressure. When applied to wood pulp having a cellulose I-type crystal structure, the reaction does not proceed inside the crystal structure. Only the primary hydroxyl group (—CH ₂ OH) of the cellulose molecular chain on the crystal surface can be selectively oxidized and converted to a carboxy group. Since electrostatic repulsion works between the carboxy groups introduced on the crystal surface, when a slight mechanical treatment is performed with the wood pulp after TEMPO oxidation dispersed in water, the cellulose fibers can reach the fine fiber unit. An aqueous dispersion of refined cellulose single nanofibers (hereinafter referred to as CSNF) can be obtained. The CSNF thus obtained has a short axis diameter as small as about 3 nm and a high aspect ratio ranging from a major axis diameter of 500 nm to several μm. A CSNF laminated film (CSNF laminated film) can be formed by applying and drying this CSNF aqueous dispersion on a substrate. The CSNF laminated film has high transparency. For example, even when a CSNF laminated film is provided on a transparent substrate to form a laminated body, the transparency can be maintained. Further, the CSNF laminated film has a structure in which highly crystalline rigid CSNF is densely accumulated, and hydrophilic carboxyl groups are densely arranged on the surface thereof, so that it has extremely high hydrophilicity (oil repellency). It is known to show.

JP-A-62-169052 Special table 2001-502959 JP 2008-1728 A

  As described above, the biomaterial storage container that has been subjected to a surface hydrophilization treatment using a water-soluble polymer such as PVA has various problems such as sample contamination and environmental load.

Accordingly, an object of the present invention is to provide a culture device that is excellent in non-adsorbability of biomaterials and is coated with a coating layer made of a material derived from a natural polymer.

The present invention for solving the above problems has the following aspects.
The present invention includes a base material and a coating layer containing finely divided cellulose fibers cross-linked by radiation irradiation, and at least a part of the base material is covered with the coating layer. .

  ADVANTAGE OF THE INVENTION According to this invention, it becomes possible to provide the environmentally friendly new plastic container for preservation | save of biomaterial, and its manufacturing method which is excellent in the non-adsorption property of a biomaterial, and there is little fear of sample contamination.

  The present invention has been made in view of the above circumstances, and is a culture apparatus characterized by providing a layer containing CSNF on the surface of various substrates or containers such as PP, for example, a biomaterial storage container, An environment-friendly new biomaterial storage container as a culture apparatus, which is excellent in non-adsorption of biomaterials due to the high hydrophilicity and oil repellency of CSNF, and has a low risk of sample contamination, and a method for producing the same provide.

≪About refined cellulose≫
The culturing apparatus of the present invention is characterized by providing a layer containing cellulose fibers, preferably CSNF, refined to the microfibril unit on the surface of various plastic substrates or containers.

  In the present invention, the number average minor axis diameter of CSNF is preferably 1 nm to 100 nm, more preferably 1 nm to 50 nm, and particularly preferably 1 nm to 20 nm. When the number average minor axis diameter is 1 nm or more, a highly crystalline finely divided cellulose fiber structure can be obtained, and the film strength of the formed coating film is good. When the number average minor axis diameter is 100 nm or less, a coating film having high transparency can be formed. Therefore, it can be suitably used as a container used for, for example, an operation performed while observing the inside.

The number average major axis diameter (average fiber length) of the CSNF is preferably 100 nm or more and 10 times or more the number average minor axis diameter. Thereby, when forming a coating film, fibers are sufficiently entangled with each other, and a sufficient film cohesive force is obtained.
The number average major axis diameter of CSNF is more preferably 200 nm or more and 100 times or more of the number average minor axis diameter.
The upper limit of the number average major axis diameter of CSNF is not particularly limited in terms of membrane cohesion, but is preferably 10 μm or less and more preferably 5 μm or less in terms of ease of production.

  The number average minor axis diameter of CSNF is obtained as an average value obtained by measuring the minor axis diameter (minimum diameter) of 100 fibers by observation with a transmission electron microscope and observation with an atomic force microscope. The number average major axis diameter of CSNF is obtained as an average value obtained by measuring the major axis diameter (maximum diameter) of 100 fibers by observation with a transmission electron microscope and observation with an atomic force microscope.

It is preferable that a carboxy group is introduced on the surface of the CSNF. The method for introducing a carboxy group is not particularly limited. For example, carboxymethylation may be carried out by reacting cellulose with monochloroacetic acid or sodium monochloroacetate in a high concentration alkaline aqueous solution. Further, a carboxy group may be introduced by directly reacting a carboxylic anhydride compound such as maleic acid or phthalic acid gasified in an autoclave with cellulose. Furthermore, a co-oxidant is used in the presence of N-oxyl compounds such as TEMPO, which has high selectivity to the oxidation of alcoholic primary carbon while maintaining the structure as much as possible under relatively mild conditions in water. The method used may be used. Oxidation reaction using an N-oxyl compound is preferable from the viewpoint of selectivity of a carboxy group introduction site and environmental load. By the oxidation reaction, carboxy groups are introduced at a high density on the fiber surface, and for example, a coating layer having extremely high hydrophilicity can be formed when applied to the substrate surface. The content of the carboxy group in CSNF can be measured by a conductivity titration method using hypochlorous acid.
In an oxidation reaction using an N-oxyl compound such as TEMPO, -CH ₂ OH at the 6th position of the glucopyranose unit of the cellulose molecular chain on the crystal surface is oxidized with high selectivity, and a carboxy group via an aldehyde group. Is converted to
Since electrostatic repulsion acts between CSNFs having a carboxy group introduced on the crystal surface in this way, reaggregation is difficult in an aqueous medium, and dispersion stability is good.
The oxidation reaction using the N-oxyl compound will be described in detail later.

  The content of the carboxy group in the CSNF is preferably in the range of 0.1 mmol or more and 5.0 mmol or less, and more preferably 0.5 mmol or more and 2.0 mmol or less per 1 g of the CSNF. When the carboxy group amount is 0.1 mmol / g or more, the dispersion stability is good. When it is 5.0 mmol / g or less, the crystal structure of CSNF is sufficiently retained, and the moldability and film cohesion are good.

In the present invention, CSNF usually contains an aqueous medium, and is used in the state of an aqueous dispersion in which the CSNF is dispersed in the aqueous medium.
As the aqueous medium, water or a mixed solution of water and an organic solvent is preferable. The organic solvent is not particularly limited as long as it is dissolved in water or mixed uniformly and does not impair the dispersibility of the modified CSNF. For example, alcohol such as methanol, ethanol, propanol, isopropanol, tetrahydrofuran, dioxane, etc. And cyclic ethers, ketones such as acetone and methyl ethyl ketone, and the like. Among these, alcohol is preferable. As the aqueous medium, water is particularly preferable.
The content of CSNF in the aqueous dispersion is not particularly limited, but is usually preferably 0.01% by mass or more and 5% by mass or less with respect to the total mass of the aqueous dispersion. If it is less than 0.01% by mass, the composition for forming a molded body becomes excessive in solvent, and it takes time to thicken the uniform coating layer to a certain thickness. If it exceeds 5% by mass, the viscosity increases due to the entanglement between CSNFs, and it becomes difficult to form a uniform coating layer.

In the present invention, the coating layer containing CSNF provided on the surface of the plastic substrate may further contain other components other than the CSNF as needed, as long as the effects of the present invention are not impaired.
As this other component, you may contain the component used for pH adjustment at the time of preparation of the aqueous dispersion of CSNF, for example. A method for preparing an aqueous dispersion of CSNF will be described in detail later.
Moreover, you may contain various additives as this other component. As an additive, it can select suitably from well-known additives according to the use etc. of the said film layer forming material. Specifically, organometallic compounds such as alkoxysilanes or hydrolysates thereof, inorganic layered compounds, inorganic needle minerals, leveling agents, antifoaming agents, water-soluble polymers, synthetic polymers, inorganic particles, organic particles , Lubricants, antistatic agents, ultraviolet absorbers, dyes, pigments, stabilizers, magnetic powders, orientation promoters, plasticizers, crosslinking agents and the like.

As the dispersion of CSNF, a dispersion produced using a known production method can be used.
Hereinafter, an example of a method for preparing a dispersion of CSNF having a carboxy group introduced from wood-based natural cellulose by an oxidation reaction using an N-oxyl compound will be described.
In this example, a wood-based natural cellulose is oxidized with an N-oxyl compound to obtain oxidized cellulose (oxidation process), and the oxidized cellulose is refined in an aqueous medium to prepare a CSNF dispersion. Including a process (miniaturization process).

(Oxidation process)
The wood-based natural cellulose is not particularly limited, and those generally used for producing cellulose nanofibers such as softwood pulp, hardwood pulp, and waste paper pulp can be used. Softwood pulp is preferred because it is easily refined and refined.
Examples of N-oxyl compounds include TEMPO (2,2,6,6-tetramethylpiperidinyl-1-oxy radical), 2,2,6,6-tetramethyl-4-hydroxypiperidine-1-oxyl, 4 -Methoxy-2,2,6,6-tetramethylpiperidine-N-oxyl, 4-ethoxy-2,2,6,6-tetramethylpiperidine-N-oxyl, 4-acetamido-2,2,6,6 -Tetramethylpiperidine-N-oxyl, etc. Among these, TEMPO is preferable.
The amount of the N-oxyl compound used is not particularly limited, and may be an amount as a catalyst. Usually, it is about 0.01-5.0 mass% with respect to solid content of the wood type natural cellulose to oxidize.

Examples of the oxidation method using an N-oxyl compound include a method in which wood-based natural cellulose is dispersed in water and oxidized in the presence of the N-oxyl compound.
At this time, it is preferable to use a co-oxidant together with the N-oxyl compound. In this case, in the reaction system, the N-oxyl compound is sequentially oxidized by a co-oxidant to produce an oxoammonium salt, and cellulose is oxidized by the oxoammonium salt. According to such oxidation treatment, the oxidation reaction proceeds smoothly even under mild conditions, and the introduction efficiency of the carboxy group is improved. When the oxidation treatment is performed under mild conditions, it is easy to maintain the crystal structure of cellulose.
As the co-oxidant, it is possible to promote an oxidation reaction such as halogen, hypohalous acid, halous acid or perhalogen acid, or a salt thereof, halogen oxide, nitrogen oxide, or peroxide. Any oxidizing agent can be used. Sodium hypochlorite is preferred because of its availability and reactivity.
The amount of the co-oxidant used is not particularly limited, and may be an amount that can promote the oxidation reaction. Usually, it is about 1-200 mass% with respect to solid content of the wood type natural cellulose to oxidize.

Along with the N-oxyl compound and the co-oxidant, at least one compound selected from the group consisting of bromide and iodide may be further used in combination. Thereby, an oxidation reaction can be advanced smoothly and the introduction efficiency of a carboxyl group can be improved.
As the compound, sodium bromide or lithium bromide is preferable, and sodium bromide is more preferable from the viewpoint of cost and stability.
The amount of the compound used is not particularly limited, and may be an amount that can promote the oxidation reaction. Usually, it is about 1-50 mass% with respect to solid content of the wood type natural cellulose to oxidize.

4-50 degreeC is preferable and, as for the reaction temperature of the said oxidation reaction, 10-40 degreeC is more preferable.
If it is 4 ° C. or lower, the reactivity of the reagent is lowered and the reaction time is prolonged. When the temperature is 50 ° C. or higher, the side reaction is promoted to lower the molecular weight of the sample, and the film cohesion force in the layer when the coating layer is formed is impaired.
The reaction time of the oxidation treatment can be appropriately set in consideration of the reaction temperature, the desired amount of carboxy group, and the like, and is not particularly limited, but is usually about 10 minutes to 5 hours.

The pH of the reaction system during the oxidation reaction is preferably 9-11. The pH is a pH at 20 ° C. When the pH is 9 or more, the reaction can be efficiently carried out. If the pH exceeds 11, side reactions may progress and the decomposition of the sample may be accelerated.
In the oxidation treatment, as the oxidation proceeds, the pH in the system decreases due to the formation of carboxy groups. It is preferable to maintain the pH of the reaction system at 9 to 11 during the oxidation treatment. Examples of a method for maintaining the pH of the reaction system at 9 to 11 include a method of adding an alkaline aqueous solution in accordance with a decrease in pH. Examples of alkaline aqueous solutions include sodium hydroxide aqueous solution, lithium hydroxide aqueous solution, potassium hydroxide aqueous solution, ammonia aqueous solution, tetramethylammonium hydroxide aqueous solution, tetraethylammonium hydroxide aqueous solution, tetrabutylammonium hydroxide aqueous solution, and benzyltrimethylammonium hydroxide aqueous solution. And organic alkalis. A sodium hydroxide aqueous solution is preferable from the viewpoint of cost.

The oxidation reaction with the N-oxyl compound can be stopped by adding alcohol to the reaction system. At this time, the pH of the reaction system is preferably maintained within the above range.
The alcohol to be added is preferably a low molecular weight alcohol such as methanol, ethanol or propanol in order to quickly terminate the reaction, and ethanol is particularly preferred from the viewpoint of safety of by-products generated by the reaction.

The reaction solution after the oxidation treatment may be directly subjected to a refinement process, but in order to remove a catalyst such as an N-oxyl compound, impurities, etc., the oxidized cellulose contained in the reaction solution is recovered and washed with a washing solution. It is preferable.
Oxidized cellulose can be collected by a known method such as filtration using a glass filter or a nylon mesh having a 20 μm pore size.
Distilled water is preferable as the cleaning liquid used for cleaning the oxidized cellulose.

(Miniaturization process)
In order to refine the oxidized cellulose, first, an aqueous medium is added to the oxidized cellulose and suspended.
Examples of the aqueous medium include those described above, and water is particularly preferable.
If necessary, the pH of the suspension may be adjusted in order to increase the dispersibility of the oxidized cellulose and the produced CSNF. Examples of the alkaline aqueous solution used for pH adjustment include the same alkaline aqueous solution as mentioned in the description of the oxidation step.

Subsequently, the suspension is subjected to physical defibrating treatment to refine the oxidized cellulose.
For physical fibrillation treatment, high pressure homogenizer, ultra high pressure homogenizer, ball mill, roll mill, cutter mill, planetary mill, jet mill, attritor, grinder, juicer mixer, homomixer, ultrasonic homogenizer, nanogenizer, underwater facing collision, etc. Mechanical treatment is mentioned.
By performing such a physical fibrillation treatment, the oxidized cellulose in the suspension is refined, and a dispersion of CSNF having a carboxy group on the fiber surface can be obtained. The number average minor axis diameter and number average major axis diameter of CSNF contained in the obtained CSNF dispersion can be adjusted by the time and number of times of physical fibrillation treatment at this time.

As described above, a dispersion of CSNF introduced with a carboxy group is obtained.
The obtained dispersion liquid can be used as a coating composition for forming a coating layer by coating on a substrate as it is, or after diluting, concentrating and the like. Moreover, you may use as a coating composition what collect | recovered CSNF contained in this coating composition, and disperse | distributed this to the dispersion medium. By using this coating composition and providing a coating layer containing CSNF on the substrate, it is possible to form an environmentally friendly coating layer that is excellent in non-adsorption of biomaterials and that does not cause sample contamination. I can do it.

The CSNF used in the present invention is not limited to that obtained by the above method.
For example, the type of cellulose used as a raw material in the above method is not limited to wood-based natural cellulose, and for example, non-wood-based natural cellulose such as cotton linter, bamboo, hemp, bagasse, kenaf, bacterial cellulose, squirt cellulose, and valonia cellulose may be used. Good. From the viewpoint of easy material procurement and stable supply, wood-based natural cellulose is preferable.
CSNF is not limited to the one obtained by the combination of the above-mentioned oxidation treatment with N-oxyl compound and physical fibrillation treatment, but chemical treatment, physical fibrillation treatment, enzyme by the oxidation treatment, dilute acid hydrolysis treatment, etc. It may be obtained by using any one of known methods such as treatment alone, or may be obtained by combining two or more. Bacterial cellulose can also be used as CSNF into which a carboxy group is introduced. In consideration of transparency and hydrophilicity when the laminate is formed, it is preferable to use CSNF obtained by refining various cellulosic materials by a combination of the above-described oxidation treatment and physical fibrillation treatment with an N-oxyl compound.

≪Culture equipment≫
The culture apparatus of the present invention has a base material and a coating layer for reducing the adsorptivity of a biomaterial on at least a part of the base material, and the coating layer contains the CSNF. It is characterized by being.

There is no restriction | limiting in particular as a base material, A well-known various sheet-like or container-like base material can be used, For example, a plastic film, a glass plate, a cellulose base material, etc. are mentioned.
Examples of the plastic material constituting the plastic film include polyolefin (polyethylene, polypropylene, etc.), polyester (polyethylene terephthalate, polyethylene naphthalate, etc.), cellulose (triacetylcellulose, diacetylcellulose, cellophane, etc.), polyamide ( 6-nylon, 6,6-nylon, etc.), acrylic (polymethyl methacrylate, etc.), polystyrene, polyvinyl chloride, polyimide, polyvinyl alcohol, polycarbonate, ethylene vinyl alcohol, and other organic polymer compounds. Also, an organic polymer material having at least one or more components from among the above-mentioned organic polymer compounds, having a copolymer component, or having a chemical modification thereof as a component is also possible. It is also possible to use bioplastics chemically synthesized from plants such as polylactic acid and biopolyolefins, plastics produced by microorganisms such as hydroxyalkanoates, and the like.
The cellulosic substrate is a substrate composed of a cellulosic material, and examples of the cellulosic material include paper, cellophane, acetylated cellulose, cellulose derivatives, and finely divided cellulose fibers.
When environmentally friendly substrates are also required due to environmental considerations, among the above, the substrates include those containing bioplastics chemically synthesized from plants and those containing plastics produced by microorganisms. A material, a cellulose base material, etc. are preferable.
The base material may contain additives such as a plasticizer, an antioxidant, a flame retardant, a filler, an antistatic agent, a crystallization accelerator, a foaming agent, a brightener, and a wettability improver.
The substrate may be subjected to surface treatment such as corona discharge, plasma treatment, oxidation treatment and the like.
Although the thickness of a base material can be suitably set according to the use etc. of the said culture apparatus, it is not specifically limited, Usually, it is about 1 micrometer-1 cm.

Formation of the coating layer containing CSNF can be carried out in the same manner as a known method. For example, an aqueous dispersion in which the modified CSNF is dispersed in an aqueous medium and optionally mixed with other components is used, and a film made of the aqueous dispersion is formed by immersing a base material in the aqueous dispersion. Is dried to form a coating layer (CSNF laminated film in which the CSNF is laminated).
The aqueous dispersion can be dried using a known drying method such as hot air drying, hot roll drying, or infrared irradiation. Although it does not specifically limit as drying conditions, As a drying temperature, 20 to 200 degreeC is preferable and 30 to 150 degreeC is more preferable. If it is 20 ° C. or less, it takes too much time to remove the aqueous medium, and if it is 200 ° C. or more, CSNF may be thermally decomposed and yellowed.
Although the thickness (thickness after drying) of a coating layer can be suitably set according to a use and is not specifically limited, 0.1-1000 micrometers is preferable and 0.2-500 micrometers is more preferable. When the thickness is 0.1 μm or more, the coating layer can be provided uniformly, and the effect of suppressing the adsorption of the biomaterial is sufficiently obtained. When the thickness is 1000 μm or less, the transparency and productivity are good.
The coating layer may be provided only on a part of the surface of the substrate, or may be provided on the entire surface.

The biological preservation container of the present invention may further have an adhesive layer (hereinafter also referred to as an anchor layer) between the base material and the coating layer.
An anchor layer is provided in order to improve the adhesiveness between a base material and a coating layer. As a method for forming the anchor layer, a known method can be used, and a two-component curable urethane resin or the like is generally used. However, the method is not limited to this, and the anchor layer can be laminated by a known method.
The thickness of the anchor layer is determined according to the purpose, but is generally in the range of 0.01 to 100 μm.

  The culture apparatus in the present invention may be subjected to a radiation irradiation process for the purpose of sterilization in the production process. Hereinafter, the effect of radiation irradiation will be described in detail.

The culture apparatus in the present invention is preferably used after sterilization, and the sterilization method is preferably radiation treatment. It has been confirmed that when the culture apparatus on which the coating layer is formed is irradiated with radiation, the structure of the coating layer changes in addition to the effect of sterilization. Specifically, CSNFs constituting the coating layer are cross-linked.
The change in the structure of the coating layer due to radiation irradiation will be described in more detail.
First, when radiation is applied to CSNF, the bonds between the atoms of cellulose molecules constituting CSNF are broken on the fiber surface of CSNF to generate radicals. Radicals are highly reactive and recombine instantly to form covalent bonds. CSNFs are cross-linked by recombination of radicals generated on the surface of adjacent CSNFs. That is, the refined cellulose fiber has a crosslinked structure.

The radiation is not particularly limited as long as radicals can be generated, but an electron beam or a gamma ray is preferable, and an electron beam is more preferable because of its excellent modification effect.
As an electron beam irradiation method, any of a curtain type, a scan type, a plasma discharge type, and the like can be applied. An acceleration voltage of the electron beam is applicable from 1 keV to 10 MeV.
Since the formation of covalent bonds is inhibited when oxygen is present in the environment where radiation is irradiated, it is preferable to irradiate the radiation in an environment where oxygen is not present. For example, during radiation irradiation, nitrogen purge or the like is preferably performed to keep the oxygen concentration at 500 ppm or less.

The radiation dose is preferably 1 kGy or more and less than 10 M Gy, more preferably 10 kGy or more and less than 8 M Gy, and particularly preferably 50 kGy or more and less than 5 M Gy.
When the irradiation dose is 1 kGy or more, a sufficient amount of covalent bonds are generated, and the effect of improving the cohesive strength of the coating layer is sufficiently obtained, and CSNF is eluted in the solvent contained in the biomaterial by cross-linking of CSNFs. The anti-contamination effect on the biomaterial can be expected. If it is less than 10 M Gy, damage to the substrate and CSNF due to radiation can be suppressed.

EXAMPLES Hereinafter, although this invention is demonstrated in detail based on an Example, the technical scope of this invention is not limited to these embodiment.
In the following examples, “%” indicates mass% (w / w%) unless otherwise specified.

[Example 1]
(TEMPO oxidation of wood cellulose)
70 g of softwood kraft pulp was suspended in 3500 g of distilled water, and a solution of 0.7 g of TEMPO and 7 g of sodium bromide dissolved in 350 g of distilled water was added and cooled to 20 ° C. 450 g of sodium hypochlorite aqueous solution having a concentration of 2 mol / L and a density of 1.15 g / mL was added dropwise thereto to initiate an oxidation reaction. The temperature in the system was always kept at 20 ° C., and the decrease in pH during the reaction was kept at pH 10 by adding a 0.5N aqueous sodium hydroxide solution. When sodium hydroxide reached 3.00 mmol / g based on the mass of cellulose, an excessive amount of ethanol was added to stop the reaction. Thereafter, filtration and washing with distilled water were repeated using a glass filter to obtain oxidized pulp.

(Measurement of carboxyl group content of oxidized pulp)
Oxidized pulp obtained by the above TEMPO oxidation was weighed by 0.1 g in terms of solid content, dispersed in water at a concentration of 1%, and hydrochloric acid was added to adjust the pH to 2.5. Thereafter, the carboxyl group amount (mmol / g) per 1 g of oxidized pulp was determined by conductivity titration using a 0.5N aqueous sodium hydroxide solution. The result was 1.6 mmol / g.

(Preparation of CSNF dispersion)
1 g of oxidized pulp obtained by the TEMPO oxidation was dispersed in 99 g of distilled water and refined with a juicer mixer for 30 minutes to obtain a CSNF aqueous dispersion having a CSNF concentration of 1%. The CSNF aqueous dispersion can be used as a coating composition in the present invention.
The number average minor axis diameter of CSNF contained in the CSNF aqueous dispersion was 3 nm, and the number average major axis diameter was 1035 nm.

(Production and evaluation of culture device)
As a substrate container, a conical tube for centrifugation (manufactured by Japan BD, 15 mL / polypropylene) was used. Plasma treatment was performed on the conical tube using a plasma treatment apparatus (MicroLab-PS, manufactured by Nissin Co., Ltd.).
The CSNF aqueous dispersion was dispensed into the tube, and the CSNF aqueous dispersion was brought into contact with the inner wall of the tube for 10 minutes. After 10 minutes, the CSNF aqueous dispersion was discharged, the tube was turned upside down, and then primary dried at 30 ° C. for 50 minutes, and then an electron beam irradiation apparatus (model LB4008, I Electron Co., Ltd.) for large substrates. Beam) was used to perform electron beam irradiation treatment so that the absorbed dose was 400 kGy. The absorbed dose was controlled by the beam current value and line speed. The acceleration voltage was fixed at 120 keV.
On the surface of the tube thus obtained, a coating layer in which CSNF was densely laminated was formed with a thickness of about 1 μm. In addition, the thickness of the coating layer was confirmed by observing a fractured surface obtained by breaking the tube in liquid nitrogen with a scanning electron microscope (S-4800, manufactured by Hitachi High-Technology Corporation).

[Example 2]
A tube was prepared in the same manner as in Example 1 except that a conical tube for centrifugation (15 mL / polystyrene, manufactured by Japan BD) was used as the substrate container.

[Comparative Example 1]
A tube was produced in the same manner as in Example 1 except that a 1% PVA aqueous solution was used instead of the CSNF dispersion.

[Comparative Example 2]
A tube was produced in the same manner as in Example 1 except that the coating layer was not formed using the CSNF dispersion.

  The following evaluation was performed about the obtained tube. Table 1 shows the evaluation items, contents, and obtained results.

(Cell recovery rate)
10 mL each of the culture solution containing rat fetal cerebral cortical neurons was dispensed into the tubes obtained in each Example and Comparative Example, and centrifuged at 1200 rpm for 10 minutes using a high-speed cooling centrifuge. . After centrifugation, the supernatant was discarded from each tube, redispersed with 10 mL of the culture solution, and then the number of cells was counted using a hemocytometer.

(Biomaterial adsorption)
-Bovine serum albumin adsorptive protein Animal serum albumin (BSAS Standard Grade (pH 7.0) Lyophilized Powder / 10 kg Container) manufactured by Veritas was used as the protein. The BSA, ¹²⁵ I-labeled by the chloramine T method, was dispersed in a phosphate buffer and diluted to 1 μg / mL. 5 mL each was dispensed into the containers obtained in each Example and Comparative Example, adsorbed onto the gel for 2 hours at 37 ° C., and then the amount of adsorption was determined by measuring the radioactivity of ¹²⁵ I.

(Storage stability)
The containers obtained in each Example and Comparative Example were placed in a constant temperature bath at 60 ° C., and the biomaterial adsorptivity evaluation was performed after storage for 30 days, and the presence or absence of deterioration of the biomaterial non-adsorption property was confirmed.

(Solvent resistance)
In each tube obtained in each example and each comparative example, 10.0 mL each of a mixed solvent of distilled water and ethanol in a ratio of 1: 1 was dispensed, and allowed to stand for 10 minutes. .

(Centrifugal strength)
The centrifugal strength was determined by visually observing deformation and cracking of each tube after centrifugation at 25,000 rpm for 10 minutes using a high-speed cooling centrifuge.

As is clear from Table 1, Examples 1 and 2 using the culture apparatus of the present invention were excellent in cell recovery rate and could suppress the adsorptivity of biological substances. Furthermore, in the storage stability test, there is no change in the adsorptivity of biological substances, and good results have been obtained for both solvent resistance and centrifugal strength, so it can be used suitably as a storage container for biological materials. It has been shown.
On the other hand, in Comparative Examples 1 and 2 in which the present invention was not used, the cell recovery rate was low, and the adsorptivity of the biological substance was also observed. In Comparative Example 2, the adsorption was particularly remarkable. In Comparative Example 1, deterioration was confirmed in the storage stability test, and the possibility of sample contamination was shown. In particular, there was a problem with solvent resistance.

The present invention can be used for a culture apparatus and a coating composition therefor.

Claims (10)

A culture apparatus comprising: a base material; and at least a coating layer containing finely divided cellulose fibers cross-linked by radiation irradiation , wherein at least a part of the base material is covered with the coating layer.   2. The culture apparatus according to claim 1, wherein the fine cellulose fiber has a number average minor axis diameter of 1 nm or more and 100 nm or less, a number average major axis diameter of 100 nm or more, and 10 times or more of the number average minor axis diameter. . The culture apparatus according to any one of claims 1 to 2, wherein a carboxy group is introduced on a surface of the fine cellulose fiber.   The culture apparatus according to claim 3, wherein the carboxy group of the fine cellulose fiber is introduced by an oxidation reaction using an N-oxyl compound. The culture apparatus according to claim 3 or 4, wherein the content of the carboxy group is in the range of 0.1 mmol to 5.0 mmol per 1 g of the fine cellulose fiber. The culture apparatus according to any one of claims 1 to 5, wherein the crystal structure of the refined cellulose fiber is cellulose I type. The culture apparatus according to any one of claims 1 to 6, wherein the biomaterial adsorption rate in the culture apparatus does not change even after storage for 30 days . A coating film coating step of coating a finely divided cellulose fiber dispersed in an aqueous medium on a substrate to form a coating film;
A coating film drying step of drying the coating film;
A radiation irradiation step of irradiating the coating film with radiation;
A method for producing a culture apparatus comprising the steps of: The method for producing a culture apparatus according to claim 8, wherein the irradiation dose of the radiation is 1 kGy or more and 10 M Gy or less. The method for producing a culture apparatus according to claim 8, wherein the radiation is an electron beam.