JPH04237494A - Nonwoven fabric for immobilizing animal cell and production thereof - Google Patents

Abstract

PURPOSE:To provide a carrier used for immobilizing animal cells and not deteriorated by autoclave sterilization treatments. CONSTITUTION:A nonwoven fabric comprising fibers consisting mainly of hydrophilic fibers is impregnated with a binder mixture comprising a hydrophilic resin binder and an animal cell-adhering material (e.g. silk fibroin, bone powder, gelatin, collagen and/or divalent cations) to combine the fibers with each other and simultaneously strongly bond the animal cell-adhering material to the fibers due to the hydrophilic resin. Since the animal cell-adhering material is dispersed in and bonded to the nonwoven fabric having a three-dimensional net structure, the nonwoven fabric has a good adhesion efficiency for animal cells and a large adhesion volume.

Description

[Detailed description of the invention]

0001

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a nonwoven fabric for immobilizing animal cells and a method for producing the same.

0002

BACKGROUND OF THE INVENTION Pharmaceutically useful physiologically active substances are produced in large quantities by culturing animal cells and the like. Animal cells used for culture include floating cells (adhesion-independent cells: an
chorage independent cel
l) and anchorage dependent cells that adhere to a suitable carrier.
ll). Since it is advantageous to use floating cells for efficient production of physiologically active substances, even when using attachment-dependent cells, there is a technique for selecting mutant strains that can grow in suspension. However, since this technique cannot be applied to all adhesion-dependent cells, a suitable carrier is required to which adhesion-dependent cells can adhere.

On the other hand, techniques for mass-producing physiologically active substances by improving cells and microorganisms using genetic recombination techniques have been put into practical use. Among these physiologically active substances, for example, proteins with sugar side chains such as vaccines and antibiotics are difficult to obtain from substances produced by microorganisms, whereas they can be obtained from substances produced by animal cells. However, since it is thought that it is easier to obtain useful physiologically active substances from animal cell-produced substances, it is expected that animal cells will be used more frequently. However, genetically modified animal cells tend to have reduced adhesion ability, and from this point of view as well, there is a desire to develop materials with excellent cell adhesion.

[0004] As a carrier for immobilizing adhesion-dependent cells,
Microcarriers have been known for a long time and have been considered particularly suitable for industrial-scale culture because they have a large specific surface area per volume. However, this carrier is not only complicated and expensive to manufacture, but also it is not always easy to attach cells to it. Furthermore, cells are damaged by the shearing force generated by agitation in the culture tank.
Care was required in handling.

[0005] Immobilization carriers prepared by coating various substrates with collagen are also known, but during autoclave sterilization performed before immobilizing animal cells, the collagen layer may run off or the collagen may Since the adhesive itself is denatured, it has the disadvantage of decreasing adhesiveness. Sterilization processes other than autoclave sterilization are known, but they require special equipment and are dangerous, so they have not been generally available.

[0006]

[Problems to be Solved by the Invention] Therefore, an object of the present invention is to provide an animal cell fixation method that can easily attach adhesion-dependent cells, is inexpensive, easy to handle, and can be sterilized using a simple autoclave. The purpose of this invention is to provide a carrier for chemical conversion.

[0007]

[Means for Solving the Problems] The present invention provides a nonwoven fabric in which constituent fibers mainly composed of hydrophilic fibers are bonded by a hydrophilic resin, wherein the animal cell adhesive material is made of the hydrophilic resin. This can be achieved by a nonwoven fabric for animal cell immobilization, which is characterized by containing in the form of a mixture with

[0008] Furthermore, the present invention is characterized in that a fibrous web mainly composed of hydrophilic fibers is impregnated with a liquid containing a hydrophilic resin and an animal cell adhesion material, and the impregnated web is dried. It also relates to a method for producing a nonwoven fabric for immobilization.

The constituent fibers of the nonwoven fabric according to the present invention are mainly hydrophilic fibers. Here, "consisting mainly" means that the constituent fibers of the nonwoven fabric contain 50% or more of hydrophilic fibers.

[0010] Any hydrophilic fiber can be used in the present invention. Hydrophilic fibers can include, for example, regenerated cellulose fibers, polyamide fibers, protein-containing fibers or natural fibers (eg cotton, linen, silk or wool). Furthermore, hydrophobic fibers (for example, polyethylene fibers, polypropylene fibers, acrylic fibers, or urethane fibers) that have been modified to be hydrophilic by any hydrophilicity imparting treatment (for example, ultraviolet irradiation or plasma treatment) are also included. This hydrophilicity imparting treatment is carried out at any stage.
In other words, even if it is carried out at the fiber or resin stage before forming the nonwoven fabric,
Alternatively, it may be carried out after forming the nonwoven fabric.

The thickness of the hydrophilic fibers used in the present invention is not particularly limited, but it is preferable to use thick fibers of 3 to 100 deniers, particularly 10 to 100 deniers. Animal cells generally have a size of about 10 to 20 μm, and are spherical in a floating state, but become flattened when attached to a carrier. Therefore, it is preferable that the fiber diameter is large because the cells can surround the fiber and flatten it. If the fiber diameter is less than 3 denier, the gaps between the fibers will become small, making it impossible for cells to enter the inside of the nonwoven fabric, which will not only reduce the cell proliferation efficiency, but also cause the nutrient components of the culture medium and dissolved substances to enter the inside of the nonwoven fabric. This is not preferable because it makes it difficult to supply oxygen, etc. On the other hand, when the fiber diameter exceeds 100 deniers, the surface area of the entire nonwoven fabric decreases and the cell adhesion efficiency decreases.
Furthermore, the processability as a nonwoven fabric is also deteriorated, which is not preferable.

In the present invention, it is preferable to use a needle-punched nonwoven fabric. Since the constituent fibers are intertwined with each other in a three-dimensional manner by the needle punching treatment, cells are easily immobilized in a state in which they enter the inside of the nonwoven fabric, and the proliferation efficiency is improved. The fiber density of the nonwoven fabric used in the present invention is 0.02 to 0.1 g/cm3.
, preferably in the range of 0.03 to 0.07 g/cm3. Fiber density is 0.02g/cm3
If it is smaller than 0.1g/cm3, it will not be able to retain its shape and the handling strength will be insufficient.On the other hand, if it is larger than 0.1g/cm3, it will not be possible to form sufficient voids in the nonwoven fabric, so cells will not be able to enter the inside of the nonwoven fabric. Not only does the number of cells decrease, but the culture solution also becomes insufficiently distributed, resulting in a decrease in the yield of the desired physiologically active substance.

In the present invention, any hydrophilic resin can be used among the resins commonly used as binders for nonwoven fabrics. The binder used in the nonwoven fabric of the present invention needs to be hydrophilic so as not to inhibit its adhesion with animal cells, and furthermore, since the nonwoven fabric with animal cells attached is used in a culture medium, it must be water insoluble. It is preferable. After the water-soluble resin is included in the constituent fibers of the nonwoven fabric, it may be made water-insoluble by heat treatment or crosslinking. Therefore,
Examples of the hydrophilic resin that can be used in the present invention include polyvinyl alcohol, cellulose, starch, and derivatives thereof. When using polyvinyl alcohol, after impregnating the constituent fibers of the nonwoven fabric with an aqueous polyvinyl alcohol solution, heat treatment (e.g., heating at about 150°C or higher) or use of a cross-linking agent (e.g., dimethyl urea) that is harmless to cells is performed. It becomes insolubilized. When polyvinyl alcohol with a high degree of saponification is used, the hydroxyl groups contained in it at a high concentration bond with the animal cell adhesive material described below through hydrogen bonds, so that the animal cell adhesive material is firmly attached to the nonwoven fabric. This is preferred because animal cells can be stably immobilized.

In the present invention, in addition to the above-mentioned hydrophilic resin,
Using specific animal cell adhesion materials. That is, the mixture of the hydrophilic resin and the specific animal cell adhesive material is used in a manner similar to that commonly used as a binder for nonwoven fabrics. The animal cell adhesion material used in the present invention is selected from the group consisting of silk fibroin, bone meal, gelatin, collagen, and divalent cation-producing salts (particularly divalent cation-producing inorganic salts).

[0015] Silk fibroin takes on a silk type II sequence when heated to the extent of autoclave treatment, and becomes a polypeptide with a stable structure. Polypeptides are inherently highly biocompatible, and silk fibroin is stabilized by heating, so it is preferred as the animal cell adhesive material of the present invention.

[0016] Bone powder is a porous body made of hydroxyapatite, and collagen is present in the pores. Therefore, it has high biocompatibility and excellent cell adhesion. Further, since the specific gravity is large, it can also be used to adjust the specific gravity of the entire nonwoven fabric for immobilizing animal cells of the present invention.

Gelatin is heat-denatured collagen and has excellent cell adhesion. If gelatin is simply coated on the constituent fibers of a nonwoven fabric, it will run off during autoclaving, but
In the present invention, gelatin is included in the constituent fibers in the form of a mixture with a hydrophilic resin, and since the gelatin is firmly attached to the constituent fibers, it does not wash away during autoclaving, and the animal cells of the present invention It can be used as an adhesive material.

Furthermore, collagen has been conventionally used as an adhesive material in carriers for immobilizing animal cells;
In the past, collagen alone was simply coated on the constituent fibers of a nonwoven fabric, which washed away during autoclaving. However, in the present invention, collagen is included in the constituent fibers in the form of a mixture with a hydrophilic resin, and since the collagen is firmly attached to the constituent fibers, it does not run off during autoclave treatment, and the present invention It can be used as an animal cell adhesive material.

[0019] Divalent cation-forming salts (particularly divalent cation-forming inorganic salts) are salts that release anions in water and become divalent cations; for example, alkali Mention may be made of the carbonates, hydrochlorides, sulphates or phosphates of earth metals (eg calcium or magnesium). When divalent cations exist on the surface of the nonwoven fabric, cells whose surface is negatively charged (-) tend to adhere to the nonwoven fabric. In particular, calcium carbonate is preferably used because it also has the effect of keeping the culture solution neutral.

[0020] In the present invention, the animal cell adhesion materials described above can be used singly or in combination of two or more.

[0021] In the nonwoven fabric for immobilizing animal cells of the present invention, the mixture of the hydrophilic resin and the animal cell adhesive material is used in the same manner as a normal binder, so that the constituent fibers of the animal cell adhesive material are mutually bonded. It can exist not only at the location where it is bonded to the fibers, but also at any location in the constituent fibers. Therefore, a structure is provided in which the animal cell adhesive material is dispersed and firmly adhered within the nonwoven fabric having a three-dimensional network structure. The amount of the animal cell adhesive material supported is not particularly limited, but in the case of silk fibroin, collagen, or gelatin, it is preferably in the range of 1 to 10 g/m2. If it is less than 1 g/m2, adhesion of animal cells will be insufficient, which is undesirable, and if it exceeds 10 g/m2, no increase in the number of animal cells will be observed as the amount carried increases. The amount of bone powder or divalent cation-producing salt supported is preferably in the range of 10 to 200 g/m2. If it is less than 10 g/m2, animal cells will not be able to adhere sufficiently, which is undesirable, and if it exceeds 200 g/m2, it will be difficult to stably support the nonwoven fabric, and furthermore, as the supported amount increases, This is not preferable because no increase in the amount of animal cells attached is observed.

[0022] Animal cells that can be immobilized on the nonwoven fabric of the present invention are not particularly limited, but for example,
Examples include cells used as hosts for genetic recombination such as BHK, L929, and CHO, and normal cells such as IMR-90 cells.

Immobilization can be carried out in the same manner as with conventional carriers for animal cell immobilization, for example, by flowing the animal cell suspension into a column packed with the nonwoven fabric for animal cell immobilization of the present invention, or by flowing the animal cell suspension into a column packed with the nonwoven fabric for animal cell immobilization of the present invention The nonwoven fabric for immobilizing animal cells of the invention may be immersed in an animal cell suspension.

The nonwoven fabric for immobilizing animal cells of the present invention is produced by, for example, impregnating a fibrous web mainly composed of hydrophilic fibers with a liquid (particularly water) containing a hydrophilic resin and an animal cell adhesive material, and It can be prepared by drying the impregnated web. The impregnation step and drying step can be carried out in the same manner as a normal nonwoven fabric manufacturing process. In addition to the hydrophilic resin and the animal cell adhesive material, the liquid used in the impregnation process may contain a crosslinking agent for the hydrophilic resin and, especially when strength is required for the nonwoven fabric, other fiber adhesive resins. I can do it. Further, the concentration of the animal cell adhesive material in the liquid can be appropriately determined depending on the type of animal cell adhesive material used. Furthermore, in the drying process, so as not to reduce the adhesive function of the animal cell adhesive material,
Although it is desirable to dry at a temperature as low as possible, in order to make the hydrophilic resin water-insoluble, it is necessary to heat it at a temperature of 150° C. or higher.

The nonwoven fabric for immobilizing animal cells of the present invention can be used as a carrier for cultured animal cells in a floating culture device or a packed culture device. In the case of a floating culture device, for example, the nonwoven fabric for immobilizing animal cells according to the present invention may be placed in a cube shape of approximately 5 mm or in a diameter of 6 mm.
The animal cells are immobilized by cutting into approximately cylindrical pieces and immersing them in an animal cell suspension. Culture is carried out in a suspended state while stirring or flowing the nonwoven fabric on which animal cells are immobilized. On the other hand, in the case of a filled-type culture device, for example, if the nonwoven fabric for immobilizing animal cells according to the present invention is filled in a culture tank as a sheet, and an animal cell suspension is passed through this, the animal cells are Fixed. After immobilizing the animal cells, the culture solution is passed through and circulated in the culture tank to culture the animal cells. In either case, the desired physiologically active substance is secreted from the cultured animal cells and can be separated and purified.

[0026]

[Examples] The present invention will now be explained in detail with reference to Examples, but these are not intended to limit the scope of the present invention.

Example 1 Rayon fiber web 170g/m2 (average fiber diameter=1
5 denier: average fiber length = 76 mm) was subjected to needle punch treatment (needle density: 200 needles/cm2) to obtain needle punch felt. Completely saponified polyvinyl alcohol (PVA-117H: Kuraray) 10% aqueous solution 50
parts by weight and 0.5 parts by weight of 1,3-dimethylurea resin and 2%
A binder mixture containing 20 parts by weight of an aqueous silk fibroin solution and made up to 100 parts by weight with distilled water was squeezed with a stainless steel mangle to a slit width of 1 mm to uniformly impregnate the needle punch felt. Cross-linked and dried at 150°C to form a carrier (fabric weight = 230 g/m
2; thickness = 3.6 mm) was obtained.

Example 2 The operation described in Example 1 was repeated, except that a 10% aqueous solution of completely saponified polyvinyl alcohol (PVA-117H: Kuraray) was used as the binder mixture.
0 parts by weight, 0.5 parts by weight of 1,3-dimethylurea resin, and 10 parts by weight of calcium carbonate.
Using the binder mixture liquid with 0 parts by weight, Example 1
Impregnation and cross-linking drying are performed in the same manner as above to obtain a carrier (fabric weight =
300g/m2; thickness=3.0mm) was obtained.

Example 3 The operation described in Example 1 was repeated, except that a 10% aqueous solution of completely saponified polyvinyl alcohol (PVA-117H: Kuraray) was used as the binder mixture.
A binder mixture containing 0 parts by weight, 0.5 parts by weight of 1,3-dimethylurea resin, and 10 parts by weight of pork bone powder (average particle size = 105 to 250 μm), and made up to 100 parts by weight with distilled water, was used. , Impregnation and crosslinking drying were performed in the same manner as in Example 1 to obtain a carrier (fabric weight = 310 g/m2; thickness = 3
．． 2 mm) was obtained.

Example 4 The operation described in Example 1 was repeated, except that a 10% aqueous solution of completely saponified polyvinyl alcohol (PVA-117H: Kuraray) was used as the binder mixture.
Using a binder mixture containing 0 parts by weight, 0.5 parts by weight of 1,3-dimethylurea resin, and 20 parts by weight of a 2% aqueous solution of gelatin (Wako Pure Chemical Industries), the total was made up to 100 parts by weight with distilled water. Impregnation and crosslinking drying were performed in the same manner as in Example 1 to obtain a carrier (fabric weight = 316 g/m2; thickness = 3.6 mm).
) was obtained.

Example 5 The operation described in Example 1 was repeated, except that a 10% aqueous solution of completely saponified polyvinyl alcohol (PVA-117H: Kuraray) was used as the binder mixture.
0 parts by weight, 0.5 parts by weight of 1,3-dimethylurea resin, 20 parts by weight of a 2% aqueous solution of silk fibroin, and 20 parts by weight of a 2% aqueous solution of gelatin (Wako Pure Chemical Industries, Ltd.).
Using a binder mixture containing 0.00 parts by weight, impregnation and crosslinking drying were performed in the same manner as in Example 1 to obtain a carrier (fabric weight = 229 g/m2; thickness = 3.3 mm).

Example 6 The operation described in Example 1 was repeated, except that a 10% aqueous solution of completely saponified polyvinyl alcohol (PVA-117H: Kuraray) was used as the binder mixture.
0 parts by weight, 0.5 parts by weight of 1,3-dimethylurea resin, 10 parts by weight of calcium carbonate, and pork bone powder (average particle size = 105~
250 μm) and 10 parts by weight, and diluted with distilled water to
Using the binder mixture liquid with 0 parts by weight, Example 1
Impregnation and cross-linking drying are performed in the same manner as above to obtain a carrier (fabric weight =
242 g/m2; thickness=2.6 mm) was obtained.

Example 7 The operation described in Example 1 was repeated, except that a 10% aqueous solution of completely saponified polyvinyl alcohol (PVA-117H: Kuraray) was used as the binder mixture.
0 parts by weight, 0.5 parts by weight of 1,3-dimethylurea resin, 20 parts by weight of 2% silk fibroin aqueous solution, 10 parts by weight of pork bone powder (average particle size = 105 to 250 μm), and 1 part by weight of calcium carbonate.
Using a binder mixture containing 0 parts by weight and 100 parts by weight with distilled water, impregnation and crosslinking drying were carried out in the same manner as in Example 1 to form a carrier (fabric weight = 310 g/m2).
; thickness = 2.0 mm) was obtained.

Example 8 The operation described in Example 1 was repeated, except that a 10% aqueous solution of completely saponified polyvinyl alcohol (PVA-117H: Kuraray) was used as the binder mixture.
0 parts by weight, 0.5 parts by weight of 1,3-dimethylurea resin, and collagen powder [Bovine: Croda Colloids Ltd.] 2
Using a binder mixture containing 100 parts by weight with distilled water, impregnation and crosslinking drying were carried out in the same manner as in Example 1 to form a carrier (fabric weight = 250 g/m2).
; thickness = 3.1 mm) was obtained.

Comparative Example 1 A polypropylene fiber web of 170 g/m 2 (average fiber diameter = 15 denier, average fiber length = 76 mm) was subjected to needle punching (needle density: 200 needles/cm 2 ) to obtain needle punch felt. Polyurethane emulsion (solid content 35%) (Merci 585: Toyo Polymer) 3
Using a binder mixture containing 0 parts by weight, 1 part by weight of an epoxy crosslinking agent (AD-C-70: Toyo Polymer) and 20 parts by weight of a 2% fibroin aqueous solution, the total was made up to 100 parts by weight with distilled water. With steel mangle,
It was squeezed with a slit width of 1 mm to uniformly impregnate the needle punch felt. It was cross-linked and dried at 150°C to obtain a carrier (fabric weight = 200 g/m2; thickness = 2.0 mm).

Comparative Example 2 The operation described in Example 1 was repeated, except that a 10% aqueous solution of completely saponified polyvinyl alcohol (PVA-117H: Kuraray) was used as the binder mixture.
Using a binder mixture containing 0 parts by weight and 0.5 parts by weight of 1,3-dimethylurea resin and made up to 100 parts by weight with distilled water, impregnation and crosslinking drying were carried out in the same manner as in Example 1 above. carrier (fabric weight = 290 g/m2; thickness = 3
．． 2 mm) was obtained.

Comparative Example 3 The needle punch felt described in Example 1 was thoroughly impregnated with a 2% aqueous solution of silk fibroin and squeezed, and the fibroin was insolubilized with absolute ethyl alcohol to form a carrier (fabric weight = 175 g/ m2; thickness = 3 mm) was obtained.

Carrier evaluation method The cell-supporting ability of each of the nonwoven fabric carriers prepared in Examples 1 to 8 and Comparative Examples 1 to 3 was evaluated by the following method. Each nonwoven fabric to be tested (15 m
m x 15 mm), sterilized in an autoclave at 121°C for 15 minutes, and cooled to room temperature. After visually evaluating the autoclavability, each test nonwoven fabric was transferred to a 12-well plate (manufactured by Corning), and a BHK cell suspension (
About 2 ml (1×10 5 cells/ml) was added. continue,
The mixture was allowed to stand for about 4 hours in air containing 5% carbon dioxide in a flan vessel at 37°C. Next, DMEM (Dulbecco
's Modified Eagle Medi
um:GIBCO) medium supplemented with 10% bovine serum and 10% tryptose phosphate broth,
It was cultured for 3 days. After evaluating the adhesion of animal cells by observing each nonwoven fabric under a microscope (200x magnification), the animal cells were peeled off by treating the test nonwoven fabric with trypsin, and the number of cells was counted using a hemocytometer. The results are shown in Table 1 below. Regarding autoclaveability in Table 1, ◯ indicates that there is almost no deterioration due to autoclaving and autoclave treatment is possible, and × indicates that there is deterioration due to autoclaving and autoclave treatment cannot be performed. Regarding cell adhesion, ◯ indicates good adhesion of animal cells, and × indicates poor adhesion of animal cells. Note that - means that the test was not conducted.

[0039]

[Table 1]

[0040]

Effects of the Invention In the nonwoven fabric for immobilizing animal cells of the present invention, an animal cell adhesive material is mixed with a hydrophilic resin binder, and the nonwoven fabric consisting of constituent fibers mainly composed of hydrophilic fibers is impregnated with the binder mixture. In addition to bonding the constituent fibers, the animal cell adhesive material is firmly attached to the constituent fibers of the nonwoven fabric using a hydrophilic resin.

[0041] Therefore, since the animal cell adhesion material having good adhesion to animal cells is contained in the form of a mixture with a hydrophilic resin and is firmly adhered to the constituent fibers of the nonwoven fabric, it can be sterilized in an autoclave. It does not run off during processing and is not easily denatured by heat. In addition, the nonwoven fabric itself does not undergo strength deterioration due to autoclave sterilization.

Furthermore, since the animal cell adhesive material is dispersed and adhered to the nonwoven fabric having a three-dimensional network structure, the animal cell adhesion efficiency is good and the animal cell adhesion capacity is large. Therefore, animal cells can be efficiently attached from a low-concentration animal cell suspension, and moreover, a large amount of animal cells can be retained and cultured. Moreover, since the nonwoven fabric for immobilizing animal cells of the present invention is composed of hydrophilic fibers and hydrophilic resin, it has good compatibility with animal cells.

In the method for producing a nonwoven fabric for immobilizing animal cells according to the present invention, the web is impregnated with the animal cell adhesive material in the form of a mixture with a hydrophilic resin, so a special process for supporting the animal cell adhesive material is required. It is not necessary and can be carried out in the same manner as a normal nonwoven fabric manufacturing method.

Claims (2)

[Claims] 1. A nonwoven fabric in which constituent fibers mainly composed of hydrophilic fibers are bonded by a hydrophilic resin, characterized in that an animal cell adhesive material is contained in the form of a mixture with the hydrophilic resin. A nonwoven fabric for immobilizing animal cells. 2. A nonwoven fabric for immobilizing animal cells, characterized in that a fibrous web mainly composed of hydrophilic fibers is impregnated with a liquid containing a hydrophilic resin and an animal cell adhesive material, and the impregnated web is dried. manufacturing method.