JPH02208330A - Yarn-like or film-like porous cellulosic material and its production - Google Patents

Abstract

PURPOSE:To obtain the title material having many interconnected cells of a specified diameter and suited as a catalyst, an adsorbent or the like by forming a cellulose (derivative) solution into a yarn or a film, freezing this yarn or film by cooling and extracting the solvent from the frozen body. CONSTITUTION:Cellulose or its derivative (e.g. cellulose acetate) is dissolved in a solvent (e.g. aqueous cuprammonium solution), and the obtained solution is formed into a yarn or a film. This yarn or film is frozen by cooling to a temperature lower than the solidification temperature of the solution, and the solvent is extracted from it or is deprived of its solvency, and the cellulose derivative, if used, is regenerated into cellulose. In this way, a yarn-like or film-like porous cellulose material having many cells of a diameter >= about 2mum, separated from each other by films and interconnected to each other through the apertures in the films which separate the adjacent cells from each other to form an interconnected porous structure, is obtained.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a filamentous or film-like porous cellulose material having a novel structure and a method for producing the same. More specifically, the present invention relates to a filamentous or film-like porous cellulose material having a structure suitable for use as a carrier for catalysts, enzymes, pharmaceuticals, ion exchangers, adsorbents, cell culture, etc., and a method for producing the same.

[Conventional technology]

Cellulose microparticles are widely used as fillers for gel filtration chromatography (GPC). In addition, because various functional groups can be easily introduced, it has a wide range of applications as a variety of ion exchangers and as a base material for affinity chromatography. Particularly in recent years, with the development of biochemistry and genetic engineering, the demand in the field of separation and purification of trace proteins in living organisms has been significantly expanding.

Furthermore, recently, techniques for culturing large amounts of high value-added proteins such as interferon or urokinase have been developed, and microcarriers for use in culturing them have been developed.

For example, Fine Chemical 1985.12.15.
For P, 5 to P, 13, chitosan porous beads (particle size 0
．． 1-3 mm, pore diameter 0.1-3 JM). Cell culture has been attempted using this microcarrier, but the cells have a relatively large diameter (for example, 10//m).
Because of this, it only attaches to the surface of the microcarrier and does not enter the pores. In this case, there is a concern that cells may fall off the surface due to collisions between microcarriers in the culture medium circulation system, resulting in a decrease in culture efficiency. In addition, microcarriers exhibit a phenomenon of floating due to the medium circulating in the culture tank, and there is a problem in that special measures are required to increase the flow rate of the medium.

As a method for immobilizing culture carriers, Japanese Patent Application Laid-Open No. 626568
Publication No. 1 discloses the use of a fibrous carrier. Examples of the fibrous carrier include glass fiber, Dacron, Teflon, nylon, and Orlon. However, these fibers are plastic fibers with a dense internal structure for clothing and industrial materials, and have a different chemical composition and structure from the fibers of the present invention. can only be expected to adhere to the surface.

Further, the well-known hollow fibers generally have a microporous structure of 0.1-= or less, and are different from the fibers of the present invention.

[Problem to be solved by the invention]

One of the uses of cellulose porous materials is as various carriers.

For example, microcarriers and fibrous carriers used for mass culture of adherent cells have conventionally been used to increase the culture concentration to 106 cells/mf by attaching cells to the particle surface, but microcarriers In case,
Because the microcarriers float in the medium, it is necessary to use a filter or sedimentation tube to separate the microcarriers and the medium supernatant, which may cause problems with filter clogging or increase the flow rate above a certain level if a sedimentation tube is used. There are problems such as not being able to do so. Further, there is also a problem that a large adhesion area cannot be obtained with a fibrous carrier.

In order to solve these problems, an immobilization carrier with a large attachment area and capable of mass culturing is desired.

SUMMARY OF THE INVENTION It is therefore an object of the present invention to solve the problems of the prior art and to
The object of the present invention is to provide a filamentous or film-like porous cellulose material in which pores larger than a voice are distributed relatively uniformly, and a method for producing the same.

[Means to solve the problem]

The above object has a large number of vacuoles separated by a membrane and having a diameter of more than about 2I1 m, and the vacuoles form a continuous pore structure that communicates with each other through openings in the membrane separating adjacent vacuoles. This is achieved by using a filamentous or film-like cellulose porous material that is characterized by the following properties:

The above-mentioned filamentous or film-like cellulose porous material is produced by forming a cellulose solution or a cellulose derivative solution into a filamentous or film-like form, cooling and freezing the solution below the solidification temperature, and then extracting and removing the solvent or removing the dissolving ability. It is produced by a method characterized by causing the cellulose derivative to be lost and, in the case of cellulose derivatives, being regenerated.

The gist of the present invention is that when a cellulose solution or a cellulose derivative solution is frozen and solidified, the solvent or its constituent components (
A type of phase separation phenomenon in which a large number of microcrystals of a solvent (hereinafter referred to as "solvent, etc.") is formed, and the dissolved cellulose or cellulose derivatives are concentrated and separated in the gaps between the solvent microcrystals is applied as a means for creating porosity. The object of the present invention is to provide a thread-like or film-like cellulose porous material (hereinafter simply referred to as "cellulose porous material") having a pore diameter larger than about 2 JJm, which has not been previously obtained, by a new method.

The size of the vacuoles of the cellulose porous material of the present invention is approximately 2 in diameter.
p, preferably the majority is 5- or more, more preferably 10-= or more. When the size is smaller than this, free movement of fluids and substances within the porous cellulose material is not achieved, and its uses are restricted. The upper limit of the size of the vacuole is not particularly limited and may be selected depending on the purpose of use and the strength of the porous cellulose material, but it is usually 500 IA.
It is often selected to be less than n, preferably less than 200 J-.

There are no particular restrictions on the thickness or structure of the vacuole septum, except that the connecting ports connecting the vacuoles to each other should be opened, but the size of the openings depends on the diameter of the vacuole. It is preferable that the diameter of the vacuole is not too small compared to the diameter of the vacuole, and preferably about 1/30 or more of the diameter of the vacuole. If the opening is too large, the strength of the cellulose porous material will be insufficient, leading to destruction during use, which is undesirable; therefore, it is desirable that the opening is about 3/4 or less, particularly about 2/3 or less, of the vacuole diameter.

The thickness of the vacuolar septum varies depending on the concentration of the cellulose or its derivative solution, the vacuole diameter, etc.
/4 or less, preferably 1/10 or less, and even 1/30 or less is possible in some cases.

In addition to the above-mentioned large-diameter openings, the diaphragm may have a finer pore structure, but this is a preferable embodiment as long as it does not particularly impede the purpose of the invention.

The cellulose porous material of the present invention, that is, the membrane constituting it,
Made essentially of cellulose.

Here, cellulose is not particularly limited and may be made from pulp, waste paper, bacterially produced cellulose, or regenerated cellulose.

The cellulose porous material used to form the cellulose porous material is dissolved and reprecipitated or regenerated by the method described below, and the average degree of polymerization is not particularly limited. The average degree of polymerization is usually preferably about 100 to 1000, but even higher degrees of polymerization, such as cellulose produced by bacteria, are a rather desirable embodiment, as long as they do not particularly impede the purpose of the invention.

Even if a small amount of hemicellulose or cellulose hydrolyzate and oxidative decomposition product is mixed in the cellulose forming the cellulose porous body, or even if a small amount of other polymer is mixed, this will defeat the purpose of the present invention. It is allowed as long as it is not.

The thread-like cellulose porous material of the present invention is a thread formed by a normal spinning method, that is, by extruding a polymer solution into a freezing medium through a discharge hole provided in a nozzle. Alternatively, it may be a hollow fiber formed by extruding a polymer solution from an annular nozzle. Furthermore, the film-like material is a film-like material formed by a normal film-forming method, that is, extruding a polymer solution through a goo or a casting method, or a film-like material formed by pouring a polymer solution into a mold. It is.

When the porous cellulose material is a thread, the cross-sectional shape is usually circular,
It is selected from polygons such as triangles and hexagons, flat polygons, flat circular shapes, hollow shapes, and box-shaped shapes, but is not limited to this range. The diameter of the thread may also be arbitrarily selected depending on the application, and it is usually 5 to 500p in diameter, and in some cases even rod-shaped threads with a diameter of 5 mm or more are possible. Needless to say, the yarn diameter does not need to be uniform in the yarn length direction, and the yarn length can also be arbitrary.

When the porous cellulose material is a film, the film thickness may be arbitrarily limited depending on the use, and is usually 5 to 500 m thick, and may even be 5 m+n or more in some cases. The film thickness does not need to be uniform, and it may be preferable to provide the film with unevenness depending on the purpose of use. Furthermore, it will be easily understood that it is also possible to take a tubular shape, a honeycomb shape, or any other arbitrary shape.

In the method of the present invention, there is no need to add foreign matter such as a porous material to the cellulose solution or cellulose derivative solution during production, so the solution has excellent moldability and a pure cellulose porous body can be easily obtained.

The diameter and shape of the vacuole are basically determined by the size and shape of crystals of the solvent, etc. that are formed when the solvent, etc. in the solution freezes and solidifies. Therefore, the shape and pore size of the vacuole can be adjusted by changing the type of cellulose solution or cellulose derivative solution and freezing assimilation conditions such as temperature.

The cellulose solution used in the present invention includes, for example, copper ammonia (Cuoxam), copper ethylenediamine (CBD),
, cadoxen, iron sodium tartrate (EWNN), nickel ethylene diamine (Nioxen), nickel ammonia (Nioxam), cobalt ethylene diamine (Cooxen), zinc ethylene diamine (Zinc)
A solution of cellulose dissolved in an aqueous solution of a metal complex such as oxen), a solution of cellulose dissolved in a dimethylacetamide/lithium chloride solvent, and various amines such as N-methylmorpholine oxide, triethylamine oxide, and cyclohexyldimethylamine. A solution of cellulose dissolved in a solvent, a solution of cellulose dissolved in a solvent containing ammonium thionate and ioar, JP-A-60-4
Examples include a solution in which cellulose is dissolved in an alkaline aqueous solution as shown in Japanese Patent No. 2438, but the present invention is not limited thereto.

The cellulose derivative solution used in the present invention includes a solution obtained by reacting paraformaldehyde with cellulose in dimethyl sulfoxide to methylolate a part of cellulose, and a solution obtained by reacting dinitrogen tetroxide with cellulose in dimethylformamide. Nitride esterified and dissolved solution, dimethyl sulfoxide (DMSO)
A solution prepared by reacting various amines and sulfur dioxide with cellulose, and a cellulose xanthate sodium solution (
viscose), and an acetone solution of cellulose acetate, but are not limited to these.

Freezing is carried out by introducing the fibrous or film solution into a medium adjusted to an arbitrary temperature. The medium is preferably a liquid or gas that is non-reactive and immiscible with the cellulose solution or cellulose derivative solution.

Note that if it is in a reactive gas or liquid, only the surface portion of the fiber or film can be reacted and modified and then frozen. For example, when frozen in a liquid or gas that is miscible with a cellulose solution or cellulose derivative solution, the liquid or gas permeates only the surface of the fiber or film before reaching the freezing temperature, so that the cellulose forms a film that covers the surface. is precipitated. As a result, a cellulose porous body whose surface layer is covered with a membrane is obtained.

When freezing is carried out in the method of the present invention, the freezing temperature is not particularly limited as long as it is lower than the temperature at which the solvent etc. freeze. However, the solvent and the like that determine the vacuole diameter of the porous cellulose material of the present invention are important in terms of crystal growth, and are selected based on the type of solvent and the desired vacuole diameter. Too low a temperature is often undesirable, as the cellulose solution will be frozen in a state close to its solution structure without forming crystals during freezing, resulting in a gel structure similar to that obtained by normal wet coagulation. However, by setting the freezing temperature appropriately, it is possible to make only the surface of the porous cellulose material have a gel structure and the inside to have a porous structure, and the surface can be partially covered with a gel film and the inside of the porous cellulose material can be partially covered with a gel film. You can also leave a connecting port. Cellulose porous materials with such a structure have particularly high resistance to deformation during compression. Generally, it is preferable that the freezing temperature is not set lower than the freezing temperature of the solvent, etc. by 40°C or more, and it is usually selected in the range of 0 to 20°C lower than the freezing temperature.

In the method of the present invention, the frozen cellulose solution or cellulose derivative solution is then treated by extracting and removing the solvent in which the cellulose or cellulose derivative is dissolved, or by lowering its solubility (hereinafter, these treatments are collectively referred to as (referred to as "solvent removal, etc.") to form a solidified cellulose porous body.

In short, the usual coagulation methods used in wet molding of cellulose solutions and cellulose derivative solutions, such as dilution precipitation or precipitation, solvent extraction, or acid-alkali neutralization reaction, can be applied as is.

Conditions such as solvent removal are not particularly limited. Normally, it is sufficient to quickly introduce the frozen filament or film into any coagulation bath or regeneration bath, but it is preferably recommended that the temperature of the coagulation bath or regeneration bath be below the freezing temperature of the solution.

However, in the case of cellulose derivatives, a cellulose regeneration step is required, and this regeneration is performed simultaneously with or sequentially with the solvent removal (that is, after the solvent removal and the like). Regeneration itself can be performed by conventional methods.

The porous cellulose material that has undergone solvent removal, etc., or the cellulose porous material that has undergone the solvent removal and regeneration process, is then washed with water or other cleaning agents, and if necessary, dried or steam sterilized. , made available for use. The washing and drying conditions are not particularly limited either, and conditions may be arbitrarily selected depending on the application.

[Function and Effects of the Invention] The porous cellulose material of the present invention has continuous vacuoles with a pore diameter larger than about 2p, so it is a structure in which liquids and solids can easily enter and exit the porous cellulose material.

The partition walls that form the vacuoles of this porous material are basically made of cellulose, which is a natural product, so they have good affinity for water, are biologically harmless, have good resistance to organic solvents, and good heat resistance. There are advantages. Because of these advantages,
Or after partial chemical modification, it is useful as a carrier for immobilized enzymes, a carrier for cell culture, etc.

Moreover, continuous vacuoles with large pores have good permeability even for viscous liquids. Furthermore, even cells with a large volume such as animal cells can enter the interior through pores on the surface, so unlike conventional surface-attached carriers, it can be used as a cell culture carrier that retains cells inside the carrier. Can be applied. Furthermore, since it is a porous material, it has a large adhesion area for cells, making it possible to culture a large amount of cells. Furthermore, due to the fibrous or film form, floating and clogging problems can also be avoided.

In addition, derivatization using the reactive hydroxyl groups of cellulose is easy, and enzyme immobilization, ion exchange ability, chelating ability, etc. can be imparted by introducing functional reactive groups, so it can be applied to various uses.

A feature of the method of the present invention is that during freezing, the solvent and the like precipitate as microcrystals, so that cellulose in the solution is concentrated at a high concentration in the small gaps between the microcrystals. In this concentrated state, cellulose is regenerated or precipitated in the form of a membrane by solvent separation, resulting in a tough cellulose porous body.

〔Example〕

Hereinafter, the present invention will be specifically described with reference to Examples.

In the examples, the viscosity of the cellulose solution or cellulose derivative solution was measured using a commercially available rotational viscometer at a temperature of 23.
This is a value measured by rotating the rotor at 2 Qrpm at ℃.

The copper ammonium relative viscosity (ηrat) of cellulose is determined by JIS P
8101, and the average degree of polymerization (U17) was determined from the copper ammonium relative viscosity using the following formula (I, E, C9
42, 502 (1950)).

(1'TF<300) 'U'F=520 ('7
rev-1) The pore diameter and pore area ratio on the surface of the porous body are:
Freeze-dried samples were subjected to gold sputtering and SE
The image was enlarged to an appropriate magnification using M and observation and measurement were performed. The diameter of the openings inside the porous body and the thickness of the diaphragm are determined by freezing the material in liquid nitrogen as described above, cutting it at the same temperature, drying it in vacuum, and then observing and measuring the particle cross section using SEM. I asked for it.

Regarding the opening diameter, etc., if the holes were not perfectly circular, they were defined as the shortest diameter.

Example 1 Dissolving pulp AL-T manufactured by Alaska Pulp Co., Ltd. was adjusted to an average degree of polymerization of 450 by acid hydrolysis, and then dissolved in an aqueous IJ solution (8% hydroxylated at 6°C) to form a cellulose solution with a concentration of 6%. Obtained.

This solution was extruded into hexane at -16° C. through a nozzle with a diameter of 1 mm to obtain a frozen filament of the solution in hexane. Next, the frozen body was taken out and put into a 50% sulfuric acid aqueous solution at -20°C, and after being kept at -20°C for 5 hours, the filamentous body was taken out and washed with water. When the filamentous bodies were observed with an optical microscope,
The thickness of the thread was 120 tones, and it was observed that many holes with a diameter of 2 to 30 μm were opened from the surface to the inside.

Furthermore, observation under high magnification revealed that the pores reached deep inside, and that each pore was separated by a membrane, forming a vacuole.

Furthermore, it was observed that the membrane separating the vacuoles formed a partially open continuous pore structure. Scanning electron microscope (SEM) photographs of the cross section and side surface of the filament are shown in FIGS. 1 and 2, respectively.

The thickness of the diaphragm determined by SEM observation of the freeze-fractured cross section was 2 feet or less.

Example 2 A film with a thickness of 1000 mm was prepared from the same cellulose solution as in Example 1 using a doctor blade, and the entire mold was immersed in silicone oil (KF96 manufactured by Shin-Etsu Silicone Co., Ltd.) cooled to -20°C. The film was removed from the mold stand midway through to complete freezing. next,
The film was taken out and put into a 50% sulfuric acid aqueous solution at -20°C, and after being kept at -20°C for 5 hours, the film was taken out and washed with water. The thickness of the obtained film was approximately 100 mm.
In 8 cases, the membrane separating the pores forms a continuous pore structure where the pores are partially open.
It was tolerated.

Example 3 This example shows one of the useful applications of the cellulose porous material of the present invention.

Cellmat, a sterilized collagen solution
rix I-Δ (manufactured by Nitta Zeradin ■) was diluted 5 times with a sterilized PT13 hydrochloric acid aqueous solution, and 0.6 mg/
Ten volumes of collagen solution were prepared and kept at 4°C.

0.4 g of the film obtained in Example 2 was collected in an undried state in terms of cellulose dry weight, placed in a pressure-resistant glass bottle with 500 mE distilled water, and sterilized in an autoclave at 130°C for 2 hours. It was kept cold at ℃. After adding 50 d of the collagen I solution described above and stirring while cooling on ice, the solution temperature was raised to 25°C over 1 hour without stirring, and the mixture was washed with sterilized water. Five recitations prepared by adding 5% fetal bovine serum (manufactured by Inu Nippon Pharmaceutical Co., Ltd.) to Ham l-12 (manufactured by Dainippon Pharmaceutical Co., Ltd.) medium were placed in a sterilized bottle with a diameter of 60 mm, and the above film was washed with water. After filling the bottle in a cylindrical shape with three layers, Chinese hamster ovary-derived cell line C11O-Kl (manufactured by Dainippon Pharmaceutical Co., Ltd.) was added and incubated at 37°C and 5% carbon dioxide for 7 days. . After incubation, the film was placed in 2% glucuraldehyde and left at 4°C for 3 hours, washed twice with phosphate buffered saline (PBS), and treated with 2% osmic acid for 1.5 hours at 4°C. . Next, 20% of 4℃,
After 50%, alcohol substitution was performed sequentially with 80%, 90%, and 100% ethanol at room temperature. After further immersing in isoamyl acetate for 30 minutes, critical point drying treatment using carbon dioxide was performed to obtain a dry sample.

This sample was subjected to gold evaporation treatment and then subjected to scanning electron microscopy (SEM).
), it was observed that Cll0-Kl had entered the pores of the film and was adhering and propagating.

[Brief explanation of the drawing]

Figures 1 and 2 respectively show a cross section and a side view of the filamentous cellulose porous material of the present invention under a scanning electron microscope (SE).
M) It is a photograph. Procedural amendment (method) % formula % Display of the case 1999 Patent Application No. 27591, Name of the invention Thread-like or film-like cellulose porous material and its manufacturing method Relationship with the case Amendment of the method Patent applicant name (003) Asahi Kasei Kogyo Co., Ltd. 4, Agent address: Shizuko Toranomon Building, 8-10 Toranomon-chome, Minato-ku, Tokyo 105 Telephone: 504-07216 Subject of amendment (1) Column 7 of "Brief explanation of drawings" in the specification; Contents of the amendment (1) On page 21, line 10 of the specification, the words ``and in Figure 2, respectively'' are corrected to ``J.'' (2) On page 21, line 11 of the specification, the words ``and'' are changed to `` Fig. 2 is a scanning electron microscope (SEM) photograph of Fig. 1.
Corrected to ``of the filamentous cellulose porous material shown in the figure.''

Claims (2)

[Claims] (1) It has a large number of vacuoles with a diameter of about 2 μm or more separated by a membrane, and the vacuoles form a continuous pore structure that communicates with each other through openings in the membrane that separate adjacent vacuoles. A filamentous or film-like cellulose porous material characterized by: (2) Form a cellulose solution or a cellulose derivative solution into a thread or film, cool and freeze the solution below its solidification temperature, then extract and remove the solvent or lose its ability to dissolve the cellulose derivative; A method for producing a filamentous or film-like porous cellulose material, which comprises regenerating the cellulose material.