JP2606213B2 - Complexes of Modified Microbial Cellulose with Gels and Animal Cell Membrane - Google Patents

Description

The present invention relates to a gel of cellulose produced by a microorganism, wherein an animal cell adhesive protein is bound to the cellulose, or the cellulose is chemically synthesized. For a gel that has been modified.

This gel is useful as a medical wound cover. In particular, a complex in which animal cells are adsorbed or bound to the gel is very useful as a medical wound cover.

[Conventional technology]

A method for mass culture by attaching animal cells to a carrier is widely used.

Conventionally used molding materials for carriers include plastic and glass as molding materials in the form of sheets or flat plates, and bead-like molding materials include cross-linked gelatin (Geri beads (KC Biological Company, USA) ) Or polyacrylamide with a charged group (such as Biocarrier (manufactured by Biorad, USA)), polystyrene (such as Biosilon (manufactured by Nunc Denmark)), dextran (Super Bees (manufactured by Flowlab, USA)), Cellulose granules (such as DE-52 (manufactured by Whatman, UK)) or dextran treated with a collagen film (such as Cytodex-3 (manufactured by Sweden, Pharmacia)) are examples of hollow fiber-shaped molding materials. Cellulose acetate (Amicon, USA)
and so on.

On the other hand, in the event of an accident or disaster, a protective material for a wound that protects the skin of a trauma or burn and promotes the regeneration of the epithelium to heal the wound is called a wound cover or an artificial skin (hereinafter referred to as “wound cover”).

Conventionally, gauze has been most widely used as a wound cover, but in recent years, freeze-dried pork skin (LPS) having better characteristics than gauze has been used. This is a biomaterial, and is excellent in that it is similar in appearance to human skin, is flexible, has good adhesion to wounds, and promotes skin regeneration.

Recently, collagen products have also appeared,
There is also a nonwoven fabric, a film, or a composite with a silicon film. It has various features such as lack of antigenicity and sterilizability.

Further, a composite of a polyamino acid film and a synthetic polymer film is being trial-produced as a wound cover. This has the feature that it is not degraded by proteases.

In addition, there are plasma membranes and fibrin membranes made from human blood. It has excellent absorbency and is not irritating to the human body.

Furthermore, as described in JP-A-59-120159, which is different from polymers and protein-based ones of these amino acids,
Some use a thin film of cellulose produced by microorganisms.

[Problems to be solved by the invention]

On the other hand, what has been conventionally used as a wound cover has the following problems. Freeze-dried pig skin and those made of collagen are easily degraded by proteases and thaw at the wound, causing bacterial infection.
I had to change frequently. The polyamino acid film is
Although it is not degraded by a protease, a suitable support such as a silicon membrane is required, and the production process is complicated. Plasma membranes and fibrin membranes made from human blood are excellent in characteristics, but the raw materials are high and supply is difficult. Cellulose thin films produced by microorganisms have problems such as poor adhesion to epithelial cells, easy peeling from wounds and easy infection of various bacteria, and not promoting skin regeneration.

Therefore, an object of the present invention is to solve the conventional problems, to provide a microorganism-produced cellulose gel which is suitable for growing animal cells and has excellent adhesion to epidermal cells.

[Means for solving the problem]

The present invention, in one aspect, is a gel of microbial-produced cellulose, wherein an animal cell adhesion protein is physically or chemically bound to the cellulose, and / or
A modified microorganism-produced cellulose gel characterized in that at least some of the hydrogen atoms in the hydroxyl groups of the cellulose are replaced by positively or negatively charged organic groups.

In another aspect, the present invention provides a complex in which animal cells are adsorbed or bound to the modified microorganism-produced cellulose gel described above.

In still another aspect, the present invention provides a complex in which animal cells are adsorbed or bound to an unmodified microorganism-produced cellulose gel.

As used herein, the term "microbial cellulose-produced gel" refers to a solid or semi-solid colloid solution comprising cellulose produced by a microorganism and a physiologically acceptable liquid (eg, distilled water, saline, glycerol). . A typical physiologically acceptable liquid is distilled water.

Microbial cellulose can be obtained as follows.

Microorganisms that produce microorganism-produced cellulose belong to the genus Acetobacter, Pseudomonas, Agrobacterium, and the like, and may be any microorganisms that produce microbial cellulose. One example is Acetobacter acetyl subsp. Xylinum (Acetobac
ter aceti subsp. xylinum) ATCC10821.

In order to produce microorganism-produced cellulose by microorganisms in the present invention, the microorganisms are inoculated and allowed to stand in a normal nutrient medium containing a carbon source, a nitrogen source, inorganic salts, and, if necessary, organic trace nutrients such as amino acids and vitamins. Alternatively, aeration and stirring may be performed gently. As the carbon source, glucose, sucrose, maltose, starch hydrolyzate, molasses and the like are used, and ethanol, acetic acid, citric acid and the like are also used alone or in combination with the other carbon sources. As the nitrogen source, ammonium salts such as ammonium sulfate, ammonium chloride and ammonium phosphate, nitrates, urea, peptone and the like are used. Amino acids as organic micronutrients,
Vitamin, fatty acid, nucleic acid, and peptone, casamino acid, yeast extract, soybean protein hydrolyzate and the like containing these are used. When using an auxotrophic mutant that requires amino acids or the like for growth, it is necessary to supplement the required nutrients. As the inorganic salts, phosphates, magnesium salts, calcium salts, iron salts, manganese salts and the like are used.

The cultivation may be performed under ordinary culturing conditions. Microbial cellulose is produced on the surface layer of the culture by culturing for 1 to 30 days while controlling the pH at 2.5 to 9 and the temperature at 20 to 40 ° C.

The microorganism-produced cellulose thus obtained has a width of 100 to 500
ÅIt has a structure in which ribbon-like microfibrils with a thickness of about 10 to 200Å are entangled, and is in a gel state containing a large amount of water.

The water content of the microorganism-produced cellulose is 95% (v / v) or more. Microbial cellulose is easily decomposed by cellulase to produce glucose. That is, a suspension of microorganism-produced cellulose 0.1 (w / v) was prepared, and cellulase (EC3.2.1.4) manufactured by Amano Pharmaceutical Co., Ltd. was 0.5%.
(W / v) in 0.1 M acetate buffer
When reacted at 0 ° C. for 24 hours, it is observed that a part of the substance is decomposed. When the supernatant is developed by paper chromatography and examined, small amounts of cellobiose, cellotriose, cellooligosaccharide and the like are detected in addition to glucose. Other small amounts of fructose, mannose and the like may be detected.

As the microorganism-produced cellulose used in the present invention, any cellulose having the above-mentioned physical properties can be used.

The microorganism-produced cellulose used in the present invention may be isolated from cultures of microorganisms or may contain some impurities. For example, residual sugars, salts,
Even if yeast extract or the like remains in the microorganism-produced cellulose, it may not. Moreover, the cells may be contained to some extent.

In addition, the cellulosic substance used in the present invention may be used as it is after washing a gel of microorganism-produced cellulose obtained by fermentation production, or the gel may be subjected to mechanical shearing force. It may be used after disintegration by acting. Any method of mechanical shearing may be used, but it can be easily performed by a rotary disintegrator, a mixer or the like.

Alternatively, the cellulosic substance in the form of a gel as it is produced by the microorganism or the disintegrated substance may be once dried and returned to the gel. The drying method is
Any method may be used, but it goes without saying that it is usually necessary to perform the reaction in a temperature range in which cellulose is not decomposed.
In addition, since the cellulosic material is composed of fine fibers having a large number of hydroxyl groups on the surface, the fibers may lose their fibrous form due to mutual adhesion during drying. Therefore, when it is desired to prevent this and make use of the fine fibrous form, it is desirable to use a method such as freeze drying or critical point drying.

The microorganism-produced cellulose gel can be complexed with an appropriate auxiliary material for the purpose of, for example, reinforcing, changing the specific gravity, immobilizing, modifying the affinity, and preventing bleeding of the liquid component. Examples of auxiliary materials to be composited include non-woven fabrics made of natural fibers such as cotton and wool, and artificial fibers such as regenerated cellulose and polyester, other fabrics, films such as polyethylene, polyvinyl alcohol, and silicone rubber, and paper. Porous film, granular material of organic or inorganic material such as alumina, glass, crystalline cellulose, agar, dextran, polyacrylamide, polyvinylpyrrolidone, alginate, chitin, hyaluronic acid, curdlan, polyacrylate, pullulan And water-soluble or polar solvents such as carrageenan, glucomannan, cellulose derivatives, and polyethylene glycol, and auxiliary materials that form a hydrophilic gel.

The complexing method is roughly classified into a method in which a substance to be complexed is put into a culture solution and cultured to produce microorganism-produced cellulose on or in the substance to be complexed. There are two methods of impregnating or backing the substance to be complexed with the microorganism-produced cellulose obtained later as a gel-like membrane, or combining the gel-like membrane after homogenizing with a homogenizer.

Animal cell-adhesive white matter is physically or chemically bound to the microorganism-produced cellulose used in the present invention, and / or the microorganism-produced cellulose is chemically modified.

As a physical binding method of proteins, there is a method in which proteins are directly adsorbed on the surface of microbial cellulose or gelled on the surface. For example, it can be adsorbed simply by immersing microorganism-produced cellulose in a protein solution. Furthermore, after adsorbing cellulose to the protein solution, the protein is gelled or solidified by changes in pH, temperature, salt concentration, metal ion concentration, etc., so that a strong physical bond can be formed. For example, specifically, in the case of collagen, the pH may be changed, in the case of gelatin, the temperature may be lowered, and when it is desired to cause cross-linking between proteins, glutaraldehyde or transglutaminase is used. You may do it.

It is also possible to increase the binding to the protein by subjecting the microorganism-produced cellulose to a known method, for example, by etherification, esterification, grafting or deoxylation.

As a chemical bonding method, a method of forming a crosslink using epihalohydrin, cyanogen halide, or the like is generally adopted. Any method may be used as long as it forms a covalent bond or a crosslink between the polysaccharide-based substance and the protein.

The protein to be bound may be any protein having the property of promoting the adhesion of animal cells to a molded article, and is generally a protein classified as a cell adhesion factor, and specifically type I, II, I
II, IV collagen, atelocollagen (trade name, Koken Co., Ltd.), fibronectin, laminin and the like.

To chemically modify microbial cellulose, the hydrogen atoms in at least some of the hydroxyl groups of the cellulose are replaced with positively or negatively charged organic groups.

For animal cell culture, it is desirable to introduce a positively charged substituent.

Substituents containing ammonia in which at least one hydrogen is substituted by alkyl, that is, the following formulas [1] and [2]
The substituent represented by is preferred.

In the formulas [1] and [2], n is an integer of 0 to 8, and R ₁
To R ₃ are a hydrogen atom or an alkyl such as methyl, ethyl and propyl, an aryl such as phenyl and naphthyl, an aralkyl such as benzene, an alkaryl, a cycloalkyl and an alkoxyalkyl group. However, R ₁ , R ₂ and R ₃ do not simultaneously represent a hydrogen atom. Also, these groups include an amino group,
A hydroxyl group or the like may be bonded.

Representative examples of the substituents represented by the formulas [1] and [2] include an aminoalkyl group (eg, an aminoethyl group), a dialkylaminoalkyl group (eg, a diethylaminoethyl group), and a trialkylaminoalkyl group (eg, triethyl Aminoethyl group), dialkylhydroxyalkylaminoalkyl group (eg, diethyl- (2-hydroxypropyl) -aluminum-ethyl group), and introduced by reacting epihalohydrin with trialcoholamine (eg, triethanolamine) and cellulose. A substituent, a quaternary halohydrin obtained by reacting an epihalohydrin with a tertiary amine, or a substituent introduced by reacting epoxide or the like containing a quaternary amine with cellulose, a guanidoalkyl group, paraamino Benzyl group, benzo Le and / or naphthoyl of dialkyl aminoalkyl group, and the like.

The positively charged substituent is not limited to the above examples as long as it can be introduced into the cellulosic substance in addition to the above. After weakly anionizing the cellulosic substance, a suitable cation such as polyethyleneimine may be adsorbed.

Examples of a method for introducing a negatively charged substituent include a method for introducing a carboxymethyl group, a carboxyethyl group, a phosphate group, and a sulfate group.

Although the modified microorganism-produced cellulose gel of the present invention can be used in various forms, it is preferably used as a sheet-like or thin-film molded product.

〔The invention's effect〕

The modified microorganism-produced cellulose of the present invention can be used as a carrier for culturing animal cells including human epidermal cells, whereby animal cells can be cultured at a high density and at a high growth rate. Unmodified microorganism-produced cellulose can also be used as a carrier for animal cell culture. Animal cells that can be cultured are cells that can adhere to the gel material of the present invention, out of all the adhesion-dependent cells of animals including humans and the adhesion-independent (suspended) cells.

Such animal cell culture can be performed using a normal cell culture medium containing 0 to 50% serum. The incubator may be an ordinary plastic dish or flask for cell culture, and if necessary, a spinner flask with a rotor or the like may be used.

The fact that human epidermal cells are cultured in a substantially monolayer state on a sheet-shaped microorganism-producing cellulose gel is very useful as a wound cover or artificial skin applied to an affected area of a burned or traumatized skin. That is, such a culture can be obtained in a relatively short time, and furthermore, it is only necessary to apply the sheet-shaped culture to the diseased part so that the cell layer is in close contact with the diseased part. Thus, the active cell layer becomes the nucleus for epidermal regeneration, and when the cellulose gel on the upper surface dries, it becomes a porous protective layer that is breathable and bacteria-impermeable. Conventionally, to prepare and apply an artificial skin analogous to this, it is necessary to culture the epidermal cells on the carrier over time until the epidermal cells become multilayered (Howard Green, Oranie Kehinde Proc. Natl. Acad. Sci. USA 1979; 76, 5665～
8), the cultured cell layer is peeled off from the carrier by a laborious operation, and the peeled surface (active cell layer side) of the multiple cell layer is applied so as to be in close contact with the affected part, and a protective material such as gauze is placed on the surface Had to be coated.

In addition, the modified microorganism-produced cellulose has high adhesion to wound tissue due to the inherent properties of the microorganism-produced cellulose and the high adhesiveness to animal cells added by the modification, so that it can be used in medical applications. Excellent as a wound covering material.

〔Example〕

 Hereinafter, examples will be described.

Example 1 Sucrose 5 g / dl, yeast extract (Difco) 0.5 g / dl, ammonium sulfate 0.5 g / dl, KH ₂ PO ₄ 0.3 g / dl, and MgSO ₄ 7H ₂ O 0.05 g / dl (pH
After 100 ml of the medium having the composition of 5.0) was steam-sterilized at 120 ° C. for 20 minutes, it was placed in a pre-sterilized petri dish having a diameter of 15 cm.

To this, acetobacter acetyl subsp. Xylinum ATCC10 grown on a test tube slope agar medium prepared by adding 2 g / dl of agar to the medium having the above composition at 30 ° C. for 7 days.
821 was inoculated in two loops and cultured at 30 ° C. Two weeks later, a gel-like film containing a cellulosic polysaccharide having a thickness of about 2 mm was formed on the upper layer of the culture solution.

After washing the membrane obtained in this way, 20% of the membrane weight 20 times
It was immersed in a NaOH solution at 100 ° C. for 30 minutes. This NaOH solution immersion operation was repeated twice more. After immersion in alkaline solution, 1NHC
After neutralizing with a solution, the gel-like membrane was washed in running water for 24 hours.

200 g of this washed milky transparent gel-like film (hereinafter referred to as “washed gel-like film”), 200 g of water and 4 g of NaOH were mixed and left at 70 ° C. for 1 hour with stirring. Further, 40 g of glycidyltrimethylammonium chloride 12 g is added to this mixture.
A solution dissolved in H ₂ O was added, and the mixture was reacted at 70 ° C. for 5 hours to perform a cationization treatment. After completion of the reaction, the alkali was neutralized with dilute hydrochloric acid, and the cationized gel film was washed with water.

The cationized gel-like membrane was cut into a circular shape having a diameter of 30 mm, and sterilized by heating in a sufficient amount of distilled water at 120 ° C. for 20 minutes. The sterilized cationized gel membrane is fully equilibrated in MEM medium containing 10% fetal calf serum (a), Dulbecco's decayed MEM medium (b), α-MEM medium (c), or RPMI medium (d) After that, it was placed in a plastic petri dish having a diameter of 30 mm. In this Petri dish (a), 4 × 10 ⁵ L-929 cells were suspended in 4 ml of MEN medium containing 10% fetal calf serum, or 2 × 10 ⁵ J-111 cells were suspended in 4 ml. 2 × 10 ^{5 in} MEM medium containing 10% fetal calf serum, Petri dish (b)
Dulbecco's modified MEM medium containing 4 ml of 10% fetal calf serum in which 3T3 Swiss albino cells were suspended, or 2 × 10 ⁵
Dulbecco's modified MEM medium containing 4 ml of 10% fetal calf serum in which M-MSV-Balb / 3T3 cells are suspended, Petri dish (c)
Medium containing 4% 10% fetal calf serum in which 2 × 10 ⁵ CHO cells were suspended, 2 × 1 in a Petri dish (d)
0 ml of 10% fetal calf serum in which ⁵ THP-1 cells were suspended was added, and the mixture was allowed to stand and cultured in a carbon dioxide gas incubator at 37 ° C for 5 days. After the cultivation, remove the cationized gel-like membrane, wash it thoroughly with phosphate buffer saline, treat it in 4 ml of 0.3% trypsin solution at 37 ° C for 20 minutes, and then use the cationized gel-like membrane using rubber policeman. The cells were detached from the surface. The number of viable cells in the cell suspension thus obtained was measured by a method using the dye excretion ability of trypan blue as an index. Table 1 shows the results.

In each case, the number was expressed by the number of viable cells per 30 mm petri dish.

Example 2 Table 2 shows the results obtained by measuring the total nitrogen content of the cationized gel-like film obtained in Example 1 by elemental analysis and the Kjeldahl method.

After drying the washed gel-like membrane, measuring the weight, and pulverizing,
Cationization was performed under the same conditions as in Example 1. Thereafter, the well was washed and dried, and the weight was measured. The results are shown in Table 3.

From the above results, it was considered that the substituent was introduced into about 0.8% of the hydroxyl groups of cellulose.

Example 3 400 ml of the medium having the composition described in Example 1 was poured into a 500 ml Sakaguchi flask, and sterilized at 120 ° C. for 30 minutes. One platinum loop of the above-mentioned Acetobacter acetyl subsp. Xylinum ATCC10821 was inoculated into each of the loops, and cultured with shaking at 30 ° C. for 20 days to obtain seeds.

The above medium (350 ml) was aseptically put into a 500 ml glass bubble column having a diameter of 4 cm, and the culture medium was added to the seeds of 50 ml and cultured at 30 ° C. for 2 weeks at aeration rate of 1/5 and VVM. Thus, a granular pellet having a diameter of 100 to 200 [mu] m is obtained.

The granular pellets were washed and subjected to a cationization treatment in the same manner as in Example 1.

The cationized granular pellets thus prepared were suspended in a sufficient amount of distilled water and sterilized by heating at 120 ° C. for 20 minutes. The sterilized cationized granular pellet was collected using a centrifuge, and suspended in a MEM medium containing 10% fetal calf serum, Dulbecco's modified MEM medium, or α-MEM medium.
After sufficient equilibration, the cationized granular pellets were collected using a centrifuge, and 8 × 10 9
⁵ was placed, 1 of 1 × 10 ⁷ cells of L-929 20 ml of the cells were suspended
MEM containing 0% fetal calf serum or 1 × 10 ⁷ J-111
A MEM medium containing 20 ml of 10% fetal calf serum in which cells are suspended or a Dulbecco's modified MEM medium containing 20 ml of 10% fetal calf serum in which 1 × 10 ⁷ M-MSV-Bilb / 3T3 cells are suspended. The mixture was stirred and cultured at 100 rpm for 5 days in the presence of carbon dioxide gas at 37 ° C. After completion of the culture, a part of the cationized granular pellet was taken, and the number of cells attached to one granular pellet was counted using an optical microscope. Table 4 shows the results.

A: Cytodex-1 (Pharmacia Co., Ltd.) using microcarriers manual (Pharmacia Co., Ltd.)
According to the above, 60 mg of cytodex-1 was suspended in 20 ml of a culture solution, 4 × 10 ⁶ cells were seeded, and the number of cells after 5 days of culture was shown.

 B shows the case where cationized granular pellets were used.

In each case, the number was expressed by the number of cells attached to the particles contained in 1 ml.

Example 4 The washed gel-like membrane obtained in Example 1 was cut into a circular shape having a diameter of 30 mm, and sterilized by heating in a sufficient amount of distilled water at 120 ° C. for 20 minutes. The sterilized washed gel membrane was neutralized with a small amount of NaOH solution, and then 1 ml of fetal calf serum and 8 ml of vitrogen
100 ml (Collagen, USA) and 1 ml of 10-fold concentrated MEM (e) or 1 ml of fetal calf serum, 8 ml of Vitrogen 100 (Colage, USA) and 1 ml of 10-fold concentrated Dulbecco's modified MEM medium And placed at 4 ° C. overnight. The gel-like membrane equilibrated with collagen was
The plate was placed in a plastic petri dish having a thickness of 10 mm and left at 37 ° C. for 10 minutes to gel collagen.

2 × 10 ⁵ L-929 cells suspended in this squall ((e) collagen-treated gel-like membrane) were suspended in 4 ml of MEM medium containing 10% fetal calf serum or 2 × 10 ⁵ J-cells. MEM medium containing 4 ml of 10% fetal calf serum in which 111 cells are suspended,
4 × 1 ml of 2 × 10 ⁵ 3T3 Swiss albino cells suspended in a Petri dish containing the collagen-treated gel membrane of (f)
0% calf 10 fetal serum were suspended in Dulbecco's modified MEM medium or 2 × 10 ⁵ pieces of M-MSV-Balb / 3T3 cells containing 4ml
3% Dulbecco's modified MEM medium containing fetal calf serum
The culture was allowed to stand still in a carbon dioxide gas incubator at 7 ° C for 5 days. After the cultivation, remove the collagen-treated gel-like membrane, wash it thoroughly with phosphate buffer saline, treat it in 4 ml of 0.3% trypsin solution at 37 ° C for 20 minutes, and then use the collagen-treated gel-like membrane with rubber policeman. The cells were detached from the surface. The number of viable cells in the cell suspension thus obtained was measured by a method using the dye excretion ability of trypan blue as an index. Table 5 shows the results.

A shows the number of cells grown on a 30 mm plastic petri dish.

 B shows the case where the washed gel film was used.

C shows the number of cells grown on the washed gel membrane treated with collagen.

 In each case, the number was expressed by the number of viable cells per 30 mm petri dish.

Example 5 200 g of the washed gel-like membrane obtained in Example 1 and water 20
0 g and 4 g of NaOH were mixed and left at 80 ° C. for 2 hours with stirring.
10 g was added over 30 minutes and reacted at 80 ° C. for 6 hours. After the completion of the reaction, the aminoethylated gel film obtained after neutralizing the alkali with dilute hydrochloric acid was washed with a sufficient amount of water.

Example 6 200 g of the washed gel-like membrane obtained in Example 1 and water 20
After 0 g and 3.5 g of NaOH were mixed and allowed to stand at 80 ° C. for 1 hour with stirring, 15 g of 2-dimethylaminoethyl chloride / hydrochloride was further added to the mixed solution together with 40 ml of water and reacted at 80 ° C. for 6 hours. After completion of the reaction, the diethylaminoethylated gel-like film obtained after neutralizing the alkali with dilute hydrochloric acid was washed with a sufficient amount of water.

Example 7 100 g of the washed gel-like film obtained in Example 1 and 10 parts of water
After 0 g and 2 g of NaOH were mixed and allowed to stand at 70 ° C. for 1 hour, 5 g of epichlorohydrin was added and left at 80 ° C. for 5 hours. After sufficiently washing the gel film after the reaction, the gel film was cut into a disk having a diameter of 30 mm, and sterilized by heating in distilled water at 120 ° C. for 20 minutes. 3 ml of Vitrogen 100 (Collagen, USA) and water
Collagen was covalently bound to the gel-like cellulosic membrane by adding 5 ml and 1 ml of 10-fold concentrated MEM and leaving it at 40 ° C. overnight.

Example 8 100 g of the washed gel-like membrane obtained in Example 1 was
In water. This was adjusted to pH 11.5 with 10 N NaOH. While gradually adding 20 g of cyanogen bromide, 10NNaOH was added dropwise so that the pH did not drop below around 11.5. The reaction was performed at 30 ° C. for 1 hour. The activated gel after the reaction was washed with running water for 3 hours. A solution prepared by dissolving 500 mg of collagen in 10 ml of water was reacted with the activated gel at 20 ° C. for 2 hours. After this reaction, the gel bound to the collagen was thoroughly washed in running water,
In Tris-HCl buffer for 24 h at room temperature to block excess cyanogen bromide. Thereafter, the Tris-HCl buffer was washed with running water.

Example 9 100 g of the washed gel-like membrane obtained in Example 1 and 100 g of water
And 3 g of NaOH, and left at 80 ° C. for 1 hour with stirring.
Next, 100 ml of a 1% aqueous solution of chloroacetic acid, the pH of which was adjusted to 12.0 with sodium hydroxide, was added to the mixture, and the mixture was reacted at 90 ° C. for 4 hours. After the reaction was completed, the alkali was neutralized with dilute hydrochloric acid, and the obtained carboxymethylated gel-like film was washed with a sufficient amount of water.

Example 10 Toxicity and pyrogen test of the washed gel membrane obtained in Example 1 900 g of the washed gel membrane obtained in Example 1
In physiological saline at 37 ° C. for 24 hours. The following test was performed using the liquid after immersion as a test liquid. As a control, physiological saline before immersion of the washed gel-like membrane was used.

Acute toxicity test A test solution was intravenously injected at a dose of 50 ml / kg to 10 mice weighing 18 to 21 g, and changes in body weight were observed every day for 5 days, but no significant difference was observed from the control.

Pyrogenic substance test The test solution was intravenously injected at a dose of 10 ml / kg to three rabbits of 1.7 to 1.9 kg, and the body temperature was measured three times at hourly intervals, but no increase in body temperature was observed.

Endotoxin Quantitative Test Endotoxin in the test solution was quantified using a colorimetric endotoxin quantification reagent Parilodic (Seikagaku Corporation), but was not detected.

Example 11 Cell culture using gel-like membrane obtained in Examples 1, 4, 5, 6, 7, 8, 9 Gel-like obtained in Examples 1, 4, 5, 6, 7, 8, 9 Membrane each 30mm in diameter
And heat sterilized in a sufficient amount of distilled water at 120 ° C. for 20 minutes or UV sterilized. After the sterilized gel membrane is fully equilibrated in MEM medium containing 10% fetal calf serum, the diameter is 30 m
m plastic dish. 4 ml of a MEM medium containing 10% fetal calf serum in which 2 × 10 ⁵ L-929 cells were suspended was put into the Petri dish, and the cells were cultured in a carbon dioxide incubator at 37 ° C. for 5 days. After cultivation,
The gel-like membrane was removed, washed well with a phosphate buffer saline, and then washed with 4 ml of a 0.3% trypsin solution.
After treatment at ℃ for 20 minutes, the cells were removed from the surface of the cationized gel-like membrane using a rubber policeman. The number of viable cells in the cell suspension thus obtained was measured by a method using the dye excretion ability of trypan blue as an index. Table 6 shows the results.

Example 12 According to the methods shown in Examples 1 and 4, a washed gel-like membrane, a cationized gel-like membrane, and a gel membrane which had been cationized and treated with collagen were aseptically obtained. Diameter each gel membrane
After shaping into a 2 cm circular shape, removing the hair on the back of a 6-week-old, male SD rat weighing 250 g, the circular epidermis of 1 to 2 cm was averaged about 0.5 to 1 mm deep from the surface with a razor blade. The experimentally created skin injury was stitched with surgical thread and fixed with gauze. Ten days later, the degree of skin regeneration at the injured part was examined.Either gel membrane was better than the petrolatum gauze used as a control, but was especially good with the cationized gel membrane and collagen treatment after cationization. When the obtained gel-like membrane was used, the adhesion to the damaged tissue was good. The gel film fell off the skin surface after 20 days.

Example 13 A washed gel membrane, a cationized gel membrane (Example 1) and a cationized gel membrane that had been treated with collagen according to the method of Example 4 were each cut into a circular shape having a diameter of 30 mm. After fully equilibrating in Dulbecco's modified MEM medium containing 5% fetal calf serum, the plate was placed in a plastic Petri dish having a diameter of 30 mm. Dullbecco's modified MEM medium containing 2 ml of 10% fetal calf serum, in which 3.5 × 10 ⁵ mouse 3T3 cells irradiated with 8000 X-ray gamma rays were suspended, was placed in this petri dish, and placed in a 37 ° C. carbon dioxide incubator. For 6 hours. After removing the medium, the method of Green et al. (Howard Green, Oranie Kehinde Proc. Natl. Acad. Sc
i. USA 1979; 79 , 5665-8) A culture medium for human epidermal keratinocytes mainly composed of Dulbecco's modified MEM medium containing 2 ml of 10% fetal calf serum in which 2 × 10 ⁵ human epidermal keratinocyte cells are suspended. (Prepared according to the method of Green et al.) And cultured in a carbon dioxide incubator at 37 ° C. for 5 days. After completion of the culture, the number of human epidermal keratinocyte cells proliferating on the gel-like membrane was measured using a microscope. Table 7 shows the results.

After the cells were substantially grown to a monolayer in the culture for 5 days as described above, the culture was further continued, and clear stratification was observed by microscopic observation 10 days later.

Example 14 A sterilized washed gel membrane, a cationized gel membrane (Example 1), and a cationized gel membrane treated with collagen according to the method of Example 4 were each cut into a circular shape having a diameter of 30 mm, and modified by Dulbecco. 30m diameter after sufficient equilibration in MEM medium
m plastic dish. A medium for human epidermal keratinocytes containing Dulbecco's modified MEM as a main component and containing 2 ml of 10% fetal calf serum in which 2 × 10 ⁵ to 4 × 10 ⁶ human epidermal keratinocytes cells are suspended in this petri dish (Green et al. Method (Howard Green Oranie Kehinde Proc. Natl. Acad. Sci. USA 1979; 79 , 5665-
Prepared according to 8). The cells were allowed to stand in a carbon dioxide gas incubator at 37 ° C. for 6 hours to allow human epidermal keratinocyte cells to adhere to the gel-like membrane. The number of human epidermal keratinocyte cells adhering on the gel-like membrane was measured using a microscope. Table 8 shows the results.

Example 15 The diethylaminoethylated gel-like film obtained in Example 6 was immersed in a 0.5% aqueous sodium alginate solution at 50 ° C. for 3 hours to thoroughly soak the sodium alginate. After removal of, a portion of the sodium alginate in the diethylaminoethylated gel-like film was gelled by immersion in a 0.1 M calcium chloride aqueous solution at room temperature for 30 minutes. After washing this, animal cells were cultured in the same manner as in Example 11, and the number of viable cells grown on the surface was 3.0 × 10 ⁶ .

Further, a diethylaminoethylated gel-like membrane which was gelled after infiltrating sodium alginate, a gel-like membrane which did not soak sodium alginate, and 0.5%
The gel strength of three calcium alginate gels obtained by gelling a 0.1% sodium alginate solution in a 0.1 M calcium chloride aqueous solution was measured with a rheometer. Table 9 shows the results.

As described above, it was found that a reinforcing effect can be obtained without influencing animal cell culture by infiltrating alginic acid into the diethylaminoethylated gel-like membrane to form a gel.

Example 16 The cationized gel-like membrane obtained in Example 1 was placed in a solution of a 7% aqueous solution of polyvinyl alcohol (average degree of polymerization: 2000).
By immersing at 24 ° C. for 24 hours, polyvinyl alcohol was well absorbed. Next, the surface was lightly washed with water to remove excess polyvinyl alcohol, and then left standing in an atmosphere at −20 ° C. for 1 hour, and then returned to room temperature to partially gel the polyvinyl alcohol. After washing this at room temperature, animal cell culture was carried out in the same manner as in Example 11, and the number of viable cells grown on the surface was 3.1 × 10 ⁶ .

In addition, only a cationized gel-like film which was gelled after infiltration of polyvinyl alcohol, and a cationized gel-like film which was not soaked and a 7% polyvinyl alcohol solution were allowed to stand in an atmosphere at -40 ° C for 1 hour. After returning to room temperature, the gel strength of the three polyvinyl alcohol gels obtained was measured with a rheometer. Table 10 shows the results.

As described above, it was found that a reinforcing effect can be obtained without influencing animal cell culture by infiltrating the cationized gel-like membrane with polyvinyl alcohol to cause gelation.

Example 17 The amount of oozing out of the liquid component in the gel was measured for a total of six types in which the gel strength was compared in Examples 15 and 16. Each gel is sandwiched between two sheets of paper
After applying a pressure of 10 g / cm ² for 10 minutes, the percentage of exudate was measured. The results are shown in Table 11.

As can be seen from Table 11, the liquid component could be prevented from oozing out by impregnating alginic acid, polyvinyl alcohol and the like into the gel-like film obtained by chemically modifying the microorganism-produced cellulose of the present invention.

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Otohiko Watanabe 1-1, Suzukicho, Kawasaki-ku, Kawasaki-shi Ajinomoto Co., Inc. (72) Inventor Hiroshiro Shirai 1-1, Suzukicho, Kawasaki-ku, Kawasaki-shi Ajinomoto (56) References JP-A-53-62889 (JP, A) JP-A-54-63894 (JP, A)

Claims (25)

(57) [Claims] 1. A gel of cellulose derived from microorganisms and having a water content of 95% (V / V) or more, wherein an animal cell adhesive protein is physically or chemically bound to the cellulose, and / or Alternatively, a modified microorganism-produced cellulose gel for use in a wound cover, wherein a hydrogen atom in at least a part of hydroxyl groups of the cellulose is replaced with a positively or negatively charged organic group. 2. The method according to claim 1, wherein the positively charged organic group is represented by the following formula [1] or [2]: (N in the formulas [1] and [2] is an integer of 0 to 8, and R ₁
~ R ₃ is a hydrogen atom or alkyl, aryl, aralkyl,
Alkaryl, cycloalkyl and alkoxyalkyl groups. However, R ₁ , R ₂ and R ₃ do not simultaneously represent a hydrogen atom. The modified microorganism-produced cellulose gel according to claim 1, wherein the gel is represented by the following formula: 3. The modified microorganism-produced cellulose gel according to claim 1, wherein the negatively charged organic group is selected from carboxymethyl, carboxyethyl, phosphate and sulfate groups. 4. The modified microorganism-produced cellulose gel according to claim 1, wherein the animal cell adhesion protein is selected from the group consisting of collagen, fibronectin, laminin and gelatin. 5. The modified microbial cellulose gel of claim 1 wherein the chemical linkage is cross-linked via epihalohydrin or cyanogen halide. 6. The method according to claim 1, wherein at least one drug selected from the group consisting of an enzyme, a bacteriostatic agent, an antibacterial agent, a chemotherapeutic agent, a coagulant and an anticoagulant is chemically or physically bound. Item 6. A gel of the modified microorganism-produced cellulose according to any one of Items 1 to 5. 7. The modified microorganism-produced cellulose gel according to any one of claims 1 to 6, wherein the auxiliary material is complexed. 8. A gel of cellulose derived from microorganisms and having a water content of 95% (V / V) or more, wherein an animal cell adhesive protein is physically or chemically bound to the cellulose, and / or Alternatively, a modified microorganism-produced cellulose gel in which hydrogen atoms in at least some of the hydroxyl groups of the cellulose are replaced with positively or negatively charged organic groups,
A complex of a modified microbial cellulose gel for use in a wound cover, wherein animal cells are bound or adsorbed. 9. The method according to claim 9, wherein the positively charged organic group is of the following formula [1] or [2]: (N in the formulas [1] and [2] is an integer of 0 to 8, and R ₁
~ R ₃ is a hydrogen atom or alkyl, aryl, aralkyl,
Alkaryl, cycloalkyl and alkoxyalkyl groups. However, R ₁ , R ₂ and R ₃ do not simultaneously represent a hydrogen atom. 9. The composite according to claim 8, which is represented by the formula: 10. The complex according to claim 8, wherein the negatively charged organic group is selected from a carboxymethyl group, a carboxyethyl group, a phosphate group and a sulfate group. 11. The complex according to claim 8, wherein the animal cell adhesion protein is selected from the group consisting of collagen, fibronectin, laminin and gelatin. 12. The conjugate according to claim 8, wherein the chemical bond is cross-linked via epihalohydrin or cyanogen halide. 13. The method according to claim 1, wherein at least one drug selected from the group consisting of enzymes, bacteriostats, antibacterial agents, chemotherapeutic agents, coagulants and anticoagulants is chemically or physically bound. 13. The conjugate according to any of paragraphs 8 to 12. 14. The complex according to claim 8, wherein the auxiliary material is further complexed with a modified microorganism-produced cellulose gel. 15. The complex according to any one of claims 8 to 14, wherein the bound or adsorbed animal cell is an adhesion-dependent animal cell. 16. The animal cell according to claim 8, wherein the animal cell bound or adsorbed is an adhesion-independent animal cell.
The conjugate according to any of the preceding items. 17. The complex according to claim 15, wherein the adhesion-dependent animal cell is a human epidermal cell. 18. The complex according to claim 17, wherein the human epidermal cells are cultured in a substantially monolayer state on a modified microorganism-produced cellulose gel. 19. A water content of 95% (V / V) derived from microorganisms.
A composite of a microorganism-produced cellulose gel used for a wound cover, wherein animal cells are bound or adsorbed to the cellulose gel. 20. At least one drug selected from the group consisting of enzymes, bacteriostats, antibacterial agents, chemotherapeutic agents, coagulants and anticoagulants is chemically or physically bound. 20. The complex according to item 19. 21. The method according to claim 21, wherein the auxiliary material is complexed with a modified microorganism-produced cellulose gel.
21. The complex according to item 19 or 20. 22. The complex according to any one of claims 19 to 21, wherein the bound or adsorbed animal cell is an adhesion-dependent animal cell. 23. The animal cell according to claim 19, wherein the animal cell bound or adsorbed is an adhesion-independent animal cell.
The conjugate according to any of the preceding items. 24. The complex according to claim 22, wherein the adhesion-dependent animal cell is a human epidermal cell. 25. The complex according to claim 24, wherein the human epidermal cells are cultured in a substantially monolayer state on a modified microorganism-produced cellulose gel.