JPH02423A - Substrate for cell culture of chitosan-collagen composite and production of the same substrate - Google Patents

Abstract

PURPOSE:To provide the title substrate of great mechanical strength where the initial conjugation and growth of cells can favorably be carried out, suitable for animal cell culture, comprising, as an active component, a chitosan-collagen composite, a combination between chitosan and collagen. CONSTITUTION:The objective substrate comprising a chitosan-collagen composite can be obtained by adding a basic aqueous solution such as ammonia water to an acidic solution of chitosan in the presence of collagen to separate a collagen out. Said chitosan is one selected from chitosan, carboxymethylchitosan and glycol chitosan. use of the chitosan-collagen acidic solution 25-90wt.% in collagen content will product the objective substrate of high cell conjugation effect.

Description

[Detailed description of the invention] [Industrial use! The present invention relates to a novel 8II2I culture substrate, and more particularly to a cell culture substrate to which cells adhere and proliferate when culturing biological cells such as animal cells or plant cells.

The cell culture substrate of the present invention can be used for cell culture of animal and plant cells.

[Description of Background of the Technology and Prior Art 1 The term "substrate for cell culture" as used herein refers to a culture medium material to which cells adhere and proliferate cells attached to the cell culture substrate in cell culture. It is a culture medium material that can be called a support material for cells in culture.

Chitosan is a polyester obtained by deacetylating chitin contained in the shells of animals such as shrimp and crabs with concentrated alkali, and contains N-acetyl-D-glucosamine and D-glucosamine.
- consists of glucosamine, but the ratio of N-acetyl-D-glucosamine and D-glucosamine in chitosan is
Depending on the degree of deacetylation, various products can be obtained.

On the other hand, collagen is a substance that is a main requirement of the connective tissue of living organisms, has excellent biocompatibility, and is widely used as a cosmetic or medical material. In addition, trobocollagen (C collagen molecule) treated with proteolytic enzymes excluding collagenase to remove the non-helical portion (C telopeptide) at the end of the molecule has very low antigenicity and is called atelocollagen.

Up to now, ready-to-use materials have been used as substrates for cell culture. For example, attempts have been made to use glass utensils, plastic petri dishes, G-decorated dextran beads, collagen beads, etc., and it has also been proposed to use single-sun beads as a substrate for tubes such as B cells. (Japanese Unexamined Patent Publication No. 62-51982) In addition, a composite material of N-acyl chitosan and collagen has been used as a biocompatible fil (I cover material, hemostatic material, or medical ITJuB for artificial organs, etc.). (Japanese Unexamined Patent Publication No. 61-253065) In cell culture, plastic petri dishes and chitosan beads have relatively fast initial adhesion of cells, but depending on the type of cells, they may not be compatible with the cells. , it may be difficult for cells to proliferate efficiently. Also, Flagenbi~
Even though they have good compatibility with #i'l+ cells, the initial adhesion of cells is slow, their mechanical strength is low, and they are expensive.

The initial adhesion of cells to the culture medium material requires fft when culturing cells using the microcarrier method, and for adhesion-dependent cells, FII is required for the culture medium material.
Since T11 growth of cells cannot be expected in the first place, it is important that the initial IS of the cells to the material of the culture medium WX is fast, and the large W of the cells is important. In the culture tube, it is desired that the initial 1gI adhesion to the material of the cell culture medium be fast. Furthermore, in order to continue the stable culture of cells, in addition to fast initial adhesion to the material of the cell culture medium, it is compatible with cells, is inexpensive and has sufficient mechanical strength. There is a demand for the development of a substrate for 7J cell culture tubes.

Mizuben 1111 and others have been conducting research on chitin and chitosan for many years.
Mechanical enough! It was found that when ff was set to 'ff L/, the initial adhesion of cells was fast, the compatibility with the cells was good, and the cells proliferated in the circle M. Based on this finding, the present invention was achieved.

[Object of the invention and summary of the invention]

The first target of the present invention is the first + of cells! The purpose of the present invention is to provide a substrate for a 1IllI culture tube that allows good 7J adhesion and cell proliferation, and more specifically, has a high mechanical strength, allows good 1TJFIJ cell adhesion and 1[II cell proliferation, The object of the present invention is to provide a thin l111rF4 tube substrate that can be suitably used for animal cell culture, and more specifically, to provide a cell culture substrate that has high mechanical strength and allows good cell adhesion and proliferation. The aim is to provide it at a low price without requiring extra costs.

The present invention provides a substrate for cell culture in which the chitosan-collagen composite has a high U effect and a high mechanical strength, and allows good initial adhesion and proliferation of A-cells.

The cell 1'3-attached substrate of the present invention can be produced by precipitating chitosan and collagen from an acidic solution of chitosan and collagen, and can be prepared by precipitating chitosan and collagen from an acidic solution of chitosan in the presence of collagen. A salt solution that can precipitate chitosan can be added to precipitate chitosan to form convexes, and a basic solution or a salt solution that can precipitate collagen can be added to the acidic solution of collagen in the presence of chitosan. va can be performed by adding and precipitating collagen.

In the production of the cell culture substrate of the chitosan-collagen composite of the present invention, the precipitation of chitosan and collagen from an acidic solution of chitosan and collagen can be achieved by removing the solution by evaporation to dryness or by adding a basic solution to acidic dissolution. This can be done by adjusting the pH, and the collagen that coexists when precipitating chitosan from an acidic solution of chitosan can be used as a collagen molded product, and collagen can be precipitated from an acidic solution of collagen. When chitosan coexists, chitosan molded products can be used, and these collagen molded products and chitosan molded products can be used to create molded products of any shape,
For example, molded products of spherical, rod-like, tubular, N-film (film and other shapes) can be used.

In the production of the cell culture substrate of the chitosan-collagen composite of the present invention, a substrate molded into a desired shape is prepared in advance, and the substrate is coated with a thin film of the chitosan-collagen composite so that the surface of the chitosan-collagen composite is coated with a thin film of the chitosan-collagen composite. It is also possible to create cell culture substrates that are composites.

Furthermore, in the production of the cell culture substrate of the present invention, by using a chitosan-collagen acidic solution containing 25 to 90% (!fl) collagen, a cell culture substrate with a high cell adhesion effect can be produced. 60～9o9
1; By using a material containing collagen (amount M), a cell culture substrate with a high cell proliferation effect can be produced.

The chitosan-collagen composite in the cell culture substrate of the present invention can be a composite in which chitosan and collagen are homogeneously mixed, or a composite in which a chitosan base is coated with a thin collagen film or a collagen base can be used. The NL/T composite can be used with a thin film of chitosan.

Furthermore, the chitosan-collagen complex in the m-cell culture substrate of the present invention can be subjected to Kuri Akebono treatment to increase its mechanical strength, and the crosslinking agent in the Kuri Jun treatment is a diisocyanate-based one. It is preferable to use

Chitosan in the cell culture substrate of the chitosan-collagen composite of the present invention can be selected from the group consisting of chitosan, carboxymethyl chitosan, glycol chitosan, and succinyl chitosan, so chitosan is understood in a narrow sense. It should be called chitosan or its derivatives.

The cell culture substrate of the present invention is easily compatible with animal cells due to the use of collagen in the chitosan-collagen complex, and the use of chitosan improves the am-strength of the cell culture substrate, and further improves the initial stage of cell culture. It is thought that this has the advantage that the adhesion speed is improved.

[Detailed description of the invention]

The chitosan-collagen complex in the cell culture substrate of the present invention is composed of a combination of chitosan and collagen, and includes a homogeneous combination of chitosan and collagen and an individual combination of chitosan and collagen. can be used.

A chitosan-collagen composite in which chitosan and collagen are homogeneously combined can be obtained by mixing an acidic solution of chitosan and an acidic solution of collagen, and evaporating the solvent from the mixed acidic solution. It can also be obtained by adding a Wg solution of a basic substance, such as aqueous ammonia, to an acidic solution and precipitating chitosan and collagen in the solution phase.

To obtain a chitosan-collagen composite molded article, a mixed acidic solution of chitosan and collagen is mixed with hydrophobic sag.
a, for example, add to toluene and stir to prepare a mixed acidic i*! of spherical chitosan and collagen! ? A system is formed in which i is dispersed in a hydrophobic organic solvent phase. Next, a solution of a basic substance, such as aqueous ammonia, is boiled in this system and stirred to precipitate chitosan and collagen in the mixed acidic solution phase of chitosan and collagen, which is taken out from the R aqueous organic solvent phase and made into beads. A shaped article can be obtained.

In addition, a mixed acidic solution of chitosan and collagen is extruded into a basic substance solution through a nozzle of a desired shape.
By precipitating chitosan and collagen, a linear or 8 m-shaped chitosan-collagen composite molded article having a cross section corresponding to the shape of the nozzle can be obtained.

Furthermore, a mixed acidic solution of chitosan and collagen is placed in a flat-bottomed container, a solution of a basic substance is added thereto, and the mixture is stirred to precipitate chitosan and collagen.
Mu ε-shaped products of the collagen complex can also be obtained.

The chitosan-collagen composite molded product of the present invention as a substrate for culturing i-cells can be of any shape, including beads, systems, holofibers (hollow m-vertebral), etc. , No! Preferably, these ε-shaped products are in the form of a chitosan-collagen composite or a film, and these ε-shaped products may be obtained by covering the surface of a substrate previously formed into a desired shape with a thin film of a chitosan-collagen composite. These substrates can be made of plastics such as polystyrene, polyester, polyamide or foams thereof, porous particles such as perlite or vermiculite, or natural fibers such as brocade or lin.

To coat these substrates with a thin film of chitosan-collagen composite, these substrates are immersed in the present tosan-collagen acidic solution, and then the chitosan-collagen complex is coated with a chitosan-collagen composite by the addition of a basic solution.
How to deposit a collagen complex on a substrate, or use chitosan-collagen acid on the substrate to reshape the container? 8M and depositing the present tosan-collagen complex on the substrate by evaporation to dryness or addition of a basic solution.

In the chitosan-collagen composite door-shaped product 119 described above, if an acidic solution of chitosan 71L or acidic Me of collagen acidic acid is used instead of the mixed acidic solution of chitosan and collagen, it is similar to the above for chitosan alone. It is possible to obtain a molded article having the shape of , or a molded article coexisting with the above-mentioned collagen electrolyte.

The chitosan-only molded article obtained in this way is immersed in an acidic solution of collagen to adhere the acidic collagen solution to the surface of the ε-shaped chitosan article. When immersed in water, collagen precipitates on a molded article made of chitosan alone, yielding an ε-shaped article of a chitosan-collagen composite in which the ε-shaped article of chitosan is covered with a 711 membrane of collagen.

Furthermore, when an ε-shaped product of collagen i is immersed in an acidic solution of chitosan and then immersed in a basic substance solution, the ε-shaped collagen product is immersed in a chitosan-collagen composite coated with a thin film of chitosan. ε-shaped products can be obtained.

When the above-described ε-shaped chitosan-collagen composite is reacted with a cross-linking agent, for example, diisocyanate, a cross-section reaction occurs in the chitosan-collagen composite, resulting in an ε-shaped chitosan-collagen composite. can be obtained.

The ε-shaped chitosan-collagen-cut Fa composite has higher R mechanical strength than the ε-shaped chitosan-collagen composite.

Chitosan, carboxymethyl chitosan, glycol chitosan, or succinyl chitosan can be used as the chitosan in the chitosan-collagen complex of the IIl cell culture substrate of the present invention, but the glycol chitosan in these chitosan derivatives is It dissolves in acidic solutions regardless of the degree of substitution. In addition, the solubility of succinyl chitosan and carboxymethyl chitosan in acidic solutions varies depending on their aa degree, so it is best to use succinyl chitosan and carboxymethyl chitosan with a low degree of substitution that has a high solubility in acidic solutions. is preferred.

Collagen neutral salt water fullerogen tissue in young animals?
Neutral transverse collagen or acid-soluble collagen obtained by extraction with 8 liquid and dilute acid aqueous solution, respectively, is used, but in these extraction procedures, insoluble collagen that is not soluble is treated with a proteolytic enzyme such as pepsin. In addition to soluble enzyme-dissolved collagen (atelocollagen), succinyl collagen or methylated collagen can also be used.

Any ratio of chitosan to collagen in the chitosan-collagen complex of the cell culture substrate of the present invention can be used;
For coating liquid, D[+, collagen is added to 5~
Preferably, it contains 50% (iJtll), and 8
For cell contact η, it is preferable to contain collagen at a ratio of 25 to 90% (C weight), and for cell proliferation, it is preferable to contain collagen at a ratio of 60 to 90% (M 71 ).

The crosslinking agent used in the crosslinking treatment of the chitosan-collagen composite of the cell culture substrate of the present invention includes diisocyanates such as hexamethylene diisocyanate, shrimp halohydrins such as epichlorohydrin, glutaraldehyde, water-soluble carbodiimide, and dicyclohexyl. Carbodiimide, Woodward's reagent or DEDQ can be used. Further, the above-mentioned crosslinking treatment can be performed by irradiating with ultraviolet rays.

The substrate for H cell @IF of the present invention is suitable for culturing animal cells, and can be used for mass culture of cells when culturing interferon, 11mm1@gen, urokinase, insulin, monoclonal antibodies, and vaccines. can.

In the following, the invention will be explained in more detail by means of examples.

Real IIIM11 (Preparation of polymer coated petri dish) 0.3% atelocollagen hydrochloric acid solution (pH: 3.Q)
and 0.3% chitosan acetic acid solution (pH: 4.0) were mixed in the proportions shown in Table 1 to prepare a polymer mixture, and the 1a7! in a polystyrene petri dish (Falcon 3
001) (diameter: 35 mm, depth: 1O order). After standing for 1 hour, the polymer mixture was drained, and the polystyrene petri dish was rinsed twice with sterile water and once with Darbetz cholic acid a solution to prepare a polymer-coated petri dish.

Table 1 Polymer mixture of chitosan and ateloco (cell adhesion test) Chicken embryo fibroblast E vesicle! Dulbecco's modified MEM medium containing 0% tallow serum was added to a concentration of 200,000 calls/-.
! JIIL/1.5- of its lfE cell suspension was injected into a polymer-coated petri dish, which was placed in a carbon dioxide gas incubator and incubated at 37°C for the time indicated in Illffl. Then, I
MIfil hang on! The number of chicken embryo SaW blast cells (K) in the cell suspension that flowed out was counted, and the number of chicken embryo wAsi blast cells (K) in the cell suspension injected into a polymer-coated Petri dish was counted. The adhesion rate (C%) of chicken embryo llam bud H follicles to each polymer-coated Petri dish was calculated by the following formula, using the difference as the number of chicken embryo*, my cells that adhered to the polymer-coated Petri dish.

Contact ratio (%) = (u-K) / (u) x l
o.

The results were as shown in Figure 1.

In the first factor, (-A-3 is sample Bl-A (chitosan:
100), (-B-) is sample 8l-8 (chitosan/atelocollagen == 75/25), (-C-) is sample Bl-C (chitosan/atelocollagen = 50/25),
50), (-D-) is sample @1-D (chitosan/atelocollagen == 25/75), (-E-3 is sample Bl-E (atelocollagen = 100), and (-0
-) is the result in an untreated polystyrene petri dish.

(Cell proliferation test) Chicken embryo msblasts were added to Dulbecco's modified HEM@III containing 10% tallow serum at 40,000 cells/1.5
- suspended at a concentration of I! 21g of the suspension was injected into a polymer-coated Petri dish (20 chicken embryo fibroblasts in the IB cell suspension), placed in a carbon dioxide culture device, and incubated at 37°C for the time shown in Figure 2. . After that, the cell suspension in the polymer-coated Petri dish was drained and washed with Dulbets' cholic acid buffer, and 0.1% trypsin-IOM EDTA buffer was added to the polymer-coated Petri dish for 10 minutes at 37°C. Incubated. After that, the proliferated chicken embryo m ostocytes were peeled off from the inner wall surface of the polymer coat, rinsed with the addition of Darbetz choline buffer solution 1tn1, and the number of chicken embryo fibroblasts (M) in the solution was counted, and the following formula was used: The proliferation rate (c%) of chicken embryo magnetoooblastic cells adhered to the polymer-coated petri dish was calculated using the following method.

Proliferation rate (%) = (2) / (U) x to.

The results were as shown in Figure 2.

In Figure 2, (-A-), (-8-1, (-C-)
, (-D-), (-E-) and (-0-) are the same as in FIG.

(Discussion) According to FIG. 1, it can be seen that chicken embryo fibroblasts adhere better to the petri dishes coated with chitosan and the chitosan-atelocollagen complex than to the untreated petri dishes and the petri dishes coated with atelocollagen.

According to Figure 2, the chitosan-atelocollagen complex,
It can be seen that in the petri dishes coated with atelocollagen and atelocollagen, chicken embryo fibroblasts exhibit a higher proliferation rate than in the untreated and chitosan coated dishes.

Example 2 (Preparation of thin film of glycol chitosan-atheloflagen composite) 1% atelocollagen salt solution pH: 3.0) 10
1 part of 0 weight f and 1% glyfur chitosan acetic acid solution (p
H: 4.0) Mix 100ffiffi parts,
Prepare a glycol chitosan-atelocollagen mixed solution, put 16 cells of it into a polystyrene petri dish (Fashion+029) (diameter: 100 mm, depth: 15 mg), air dry, and place the glycol chitosan-atelocollagen complex on the inner bottom of the polystyrene petri dish. The thin film layer was made of cedar bark. The thin film layer of this Glyfur chitosan-atelocollagen complex was peeled off from the polystyrene petri dish, immersed in a methanol-ammonia (4:I) solution to deoxidize it, and thoroughly washed with methanol. A thin film of the composite was immersed in methanol containing 2% hexamethylene diisocyanate, and a crosslinking reaction was carried out for 2 hours. This thin film of the glycol chitosan-atelocollagen scaffold 1 composite was thoroughly washed with methanol and then thoroughly washed with water to prepare a thin film of the glycol chitosan-atelocollagen scaffold composite.

(Cell adhesion test) A thin film of the glycol chitosan-athelocollagen crosslinked composite was placed in a polystyrene Petri dish (Fashion 300).
2) (diameter = 60mm, depth: 15++g) and a glass ring (outer diameter: 45mm, inner diameter: 25mm) on top of the thin film.
11j, height: 5IIj+) was placed in (-77), and the chicken #5 fibroblast IB cells were suspended in Dulbecco's modified HEM@i11 containing lθ% beef tallow serum at a rate of 200,000 calls/i. Inject 0.75 mm of the cloudy cyst suspension, place the polystyrene petri dish in a carbon dioxide culture device, and incubate at 37°C.
The cells were incubated for the time shown in FIG. Upper aF
Remove the Fl, and remove the supernatant from the chicken embryo m-cone bud li [! The number of follicles (K) was counted, and the number of chicken embryo m1m buds 81a (
U) m (U-K)'E-g IJ: As the number of chicken embryo filamentous buds that adhered to the NI membrane of the znm combination of 1-luchitosan-atheloflagen, the adhesion rate (%) was calculated using the following formula: Calculated by.

Tangent tJ (%) = (u-K) / (tl) x
to.

1% atelocollagen hydrochloric acid solution H (pH: 3.0) was used, and 1% glycol chitosan vinegar solution (pH: 4.0) was used.
A thin film of Kurihashi bodies of atelocollagen was prepared in the same manner as described above without using 0), and a thin film of Kurihashi bodies of atelocollagen was used to adhere the chicken osteoblasts in the same manner as above. rate c%) was calculated.

Furthermore, without using a thin film of glycol chitosan-atelocollagen composite, a glass ring (outer diameter: 45 s+m, inner diameter: 251111, height: 5jL11) was placed in a polystyrene petri dish (Fashion 3002).
), and the adhesion rate (%) of chicken embryo fibroblasts was calculated in the same manner as above.

These results were as shown in FIG.

In Figure 3, (-〇-) is glycol chitosan-
The thin film of the atelocollagen scaffold complex, (-X-) indicates the Wl membrane of the atelocollagen scaffold, and (-Δ-) indicates the adhesion rate of chicken embryo fibroblasts in a polystyrene petri dish (Fashion 3002).

(Cell proliferation test) A thin film of the glycol chitosan-athelocollagen crosslinked complex was placed in a polystyrene Petri dish (Faltocon 30
02n ζ diameter: 60 jlJI, depth: 15IIg
) and a glass ring (outer diameter: 45 mm) on top of the thin film.
Me, inner diameter: 251111. Height: 5++x)
Chicken embryo fibroblasts were placed in Dulbecco's modified MEN medium containing 10% tallow serum at 20,000 cells/0.
A cell suspension suspended at a concentration of 0.75- was injected into a polystyrene petri dish, and the number of chicken embryos and male buds in the cell suspension (mW) was U), and the polystyrene petri dish was placed in a carbon dioxide culture device. 4th cup at 37℃
Incubate for the times indicated.

After removing the supernatant, the thin film of the glycol chitosan-athelocollagen cross-linked complex in the polystyrene petri dish was washed with Dulbets choline buffer 1d, and the concentration of 0.196
Tribucin-10mM EDTA solution 1, 3
Incubated for 10 minutes at 7"C. Then
Chick embryo fibroblasts grown from a thin film of a glycol chitosan-atelocollagen crosslinked complex were stripped, washed with Dulbets' cholic acid buffer, and the number of chicken embryo fibroblasts (M) in the washing solution was counted; The proliferation rate (C%) of chicken embryo amoblasts adhering to the thin film of the glycol chitosan-atelocollagen crosslinked complex was calculated using the following formula.

Increase M rate c%) = (M) / (u) X ro.

Using 1% atelocollagen salt #Keiju (11H: 3.0), 1% glycol chitosan acetic acid solution (1)H:
A thin film of cross-linked atelocollagen was prepared in the same manner as above without using 4.0), and using this thin film of atelocollagen, the proliferation rate (% calculated.

Furthermore, without using a thin film of the glycol chitosan-atheloflagen crosslinker, a glass ring (outer diameter: 45 mm) was placed directly on a polystyrene Petri dish (Methylcon 3002).
xIm, inner diameter: 25m11+, height: 5mm) was placed, and in the same manner as above, N increase rate (%) of chicken embryo m pyramidal cells was determined.
was calculated.

These results were as shown in FIG.

In Fig. 4, (-0-) is a thin film of a cross-linked complex of glycol chitosan and atelocollagen, (-X-) is a thin film of a cross-linked atelocollagen complex, and C-△-) is a polystyrene petri dish (Methylcon 3002). Figure 2 shows the N increase rate of chicken embryo fibroblasts in .

(Discussion) According to Figures 3 and 4, glycol chitosan-
It can be seen that the atelocollagen crosslinked complex and the atelocollagen crosslinked body exhibit poorer cell adhesion rates and liii cell proliferation rates than those without these.

Example 3 (TIIWJ of thin film of crosslinked composite of carboxymethyl chitosan-atelocollagen) 1% atelocollagen hydrochloric acid solution (pH: 3.0) 10
0! The fit part was treated with 1% carboxymethyl chitosan (degree of substitution of carboxymethyl group: O, 15) acetic acid solution (pH:
4.5) Mix 200 parts by weight and make the mixture 16a
11 in a polystyrene Petri dish (Methylcon 1029)
(diameter: 100 m, depth: 15 u) and left to stand to air dry to form a thin film layer of carboxymethyl chitosan-athelocollagen complex on a polystyrene petri dish. The thin film of this carboxymethyl chitosan-atelocollagen composite was peeled off from the polystyrene petri dish, and after exposing the thin film to an ammonia gas atmosphere for 2 hours, it was thoroughly washed with water, and the thin film was soaked in a 1% carbodiimide aqueous solution for 3 hours.
At 06C, a thin film of a carboxymethyl chitosan-athelocollagen crosslinked composite was prepared by soaking overnight, reacting, and thoroughly washing with water.

(Cell adhesion test) Glycol chitosan in the cell adhesion test of Example 2
An NI cell adhesion test was conducted in the same manner as in Example 2, using the above thin film of the crosslinked carboxymethyl chitosan-atelocollagen complex instead of the thin film of the crosslinked atelocollagen complex.

The results were as shown in FIG.

In Fig. 5, (-〇-) is a thin film of a cross-linked composite of carboxymethyl chitosan-athelocollagen, (-X-)
is a thin film of crosslinked atelocollagen, and (−Δ−)
The figure shows the adhesion of chicken fibers in a polystyrene Petri dish (Methylcon 3002).

(Cell proliferation test) Glycol chitosan in the cell proliferation test of Example 2
An MI cell proliferation test was conducted in the same manner as in Example 2, using the above thin film of the crosslinked carboxymethyl chitosan-atelocollagen composite instead of the thin film of the crosslinked atelocollagen composite.

The results were as shown in Figure 6.

In Fig. 6, (-0-) is a thin film of chestnut body of carboxymethyl chitosan-atelocollagen, (-X-)
is a thin film of the atelocollagen bridge body, and (-△-)
Figure 1 shows the growth of chicken embryo fibroblasts in polystyrene petri dishes (Methylcon 3002).

(Consideration W) According to Figures 5 and 6, the carboxymethyl chitosan-atelocollagen cross-Fs complex is as good as a single atelocollagen fruit! ! P#:X and cell proliferation rate of 1 indicates that the carboxymethyl chitosan-atelocollagen composite is an excellent substrate for cell culture.

Implementation (Preparation of 4C succinyl chitosan-atelocollagen cross-body composite beads) uft 50 g of 3% atelocollagen hydrochloric acid solution with 150 g of 3% succinyl chitosan (succinylation g: 0.2) in acetic acid, and prepare succinyl chitosan-atelocollagen. A mixed lysis was prepared.

Il! Put 400 yd of toluene and chloroform+10- in a volume heater, add Span 20 to the mixed solvent, and prepare 0.1% (mI21) noaMt7)7. A mixed solvent containing Han 20 was prepared, and to this mixed solvent was added the previously prepared succinyl-atelocollagen mixed solution + 50
-, stir vigorously to form a spherical succinylchitosan-atelocollagen mixed solution phase in the mixed medium phase, and add ammonia water while stirring to precipitate the succinylchitosan-atelocollagen complex. After forming succinyl chitosan-atelocollagen composite beads, the mixed solvent was decanted from the beaker, and the succinylchitosan-atelocollagen composite beads were thoroughly washed with methanol. These succinyl chitosan-atelocollagen composite beads were mixed with a 1% carbodiimide aqueous solution.
00- and stirred to disperse the succinyl chitosan-athelocollagen composite beads in the liquid, and stirred overnight at 30°C to form succinyl chitosan-athelocollagen composite beads.
After the atelocollagen chestnut Akebono composite beads were taken out from the liquid, they were thoroughly washed with water, and the 48 to 100 mesh fractions were classified to prepare succinyl chitosan-athelocollagen chestnut 1 composite beads.

(Fine @ Adhesion and Cell Proliferation Test) Mouse skin-derived w4 androblasts (Lllllllll) were suspended in MEN F medium containing 10% live fetal serum, and then succinylchitosan-athelocollagen chestnut FIJta was added to the cell suspension solution containing 10% live fetal serum. The combined beads were placed in a carbon dioxide incubator and incubated at 37°C for 5 days. The surface of the succinyl chitosan-athelocollagen scaffold 1 composite beads was sometimes covered with proliferated cells.

Implementation @ 5 (Preparation of chitosan-atelocollagen composite holofiber) 2% atelocollagen in hydrochloric acid El (pH: 3.0)
50g of 2% chitosan acetic acid solution (pu: 4.0)
50y to prepare a chitosan-athelocollagen mixture m liquid, and this chitosan-athelocollagen mixture G
The liquid was discharged with an external inner diameter of 71.5 ms, a circular node outer diameter: Q,
Holofibers were formed by extrusion through a 7 gm annular nozzle into a 35% sodium chloride aqueous solution bath at a speed of 4 m/min. In this extrusion, a 35% aqueous sodium chloride solution was discharged from the inner cylinder of the annular nozzle to form an internal liquid.

The coagulated holofiber (medium-!2! thread) was irradiated with a 30W ultraviolet lamp at the zero point of 20-, and then 5%
Immerse for 5 minutes in a 0.1% aqueous sodium carbonate solution containing 1 to 1% sodium chloride, and then rinse with running water for 10 minutes in a washing tank.
After washing with water for a minute, it was dried at 40° C. while blowing air from one end of the capillary to prepare a chitosan-athelocollagen composite holofiber.

(Cell adhesion and cell proliferation test) Cell adhesion and cell proliferation tests were conducted in the same manner as in Example 4 using the above chitosan-atelocollagen composite holofiber. In that test, chitosan-
Atelocollagen composite holofiber is IIJI!, which is derived from similarly grown mouse skin. It was mostly covered with blast cells.

Example 6 (Preparation of chitosan-coated collagen beads) Commercially available collagen beads (Koken Celgen Microsphere) 30- were added to 0.3% chitosan solution 10011i, stirred slowly to disperse, and then the collagen beads were filtered out. The collagen beads were placed in a 5% ammonia aqueous solution to precipitate chitosan on the surface of the collagen beads. After washing the collagen beads with water and methanol,
The collagen beads to which chitosan was attached were immersed in a 2% hexamethylene diisocyanate solution and reacted for 2 hours at 20°C. The beads were filtered and washed sequentially with methanol and water to prepare chitosan-coated collagen beads.

(Cell Adhesion and Cell Proliferation Test) Cell adhesion and cell proliferation tests on mouse skin-derived fibroblasts were conducted in the same manner as in Example 4 using the chitosan-coated collagen beads described above.

In that test, chitosan-coated collagen beads were substantially covered with proliferated mouse skin-derived fibroblasts.

Example 7 (Preparation of atelocollagen-coated chitosan beads) II
! Toluene 400- and chloroform +
10- is added, Span 20 is added to the mixed solvent, and 0.
Prepare a mixed solvent containing Span 20 with a concentration of 1% (!Ii), add 3% chitosan acetic acid solution 150- to this mixed solvent, and stir vigorously to form a spherical chitosan liquid phase in the mixed solvent phase. After aqueous ammonia was formed and stirred, chitosan was precipitated to form chitosan beads, the mixed C medium was decanted from the beaker, and the chitosan beads were thoroughly washed with methanol.

In a 300-capacity beaker, put 0.3% atelocollagen salt hydrochloric acid solution pH: 3.0) +00-, add the chitosan beads 30- obtained above, stir slowly to disperse, and then add the chitosan beads. were separated by filtration, and the chitosan beads were placed in 5% ammonia water to precipitate atelocollagen on the surface of the chitosan beads. After washing the chitosan beads on which atelocollagen was precipitated with water and thoroughly washing with methanol, the chitosan beads on which atelocollagen was precipitated were immersed in a 2% hexamethylene diisocyanate methanol solution, allowed to cross-react for 2 hours, and the beads were soaked in methanol. The beads were thoroughly washed with water to obtain atelocollagen-coated chitosan beads.

C Cell Adhesion and Cell Proliferation Test) Cell adhesion and cell proliferation tests of mouse skin-derived fibroblasts (L cells) were conducted in the same manner as in Example 6 using the atelocollagen-coated chitosan beads described above.

In that test, atelocollagen-coated chitosan beads were covered with proliferated mouse skin-derived amoblasts.

Example 8 (Preparation of thin film of chitosan-atelocollagen complex) 1% (V/V) atelocollagen hydrochloric acid solution (pH: 3
．． 0) and 1% (-t/V) chitosan acetic acid solution [acetic acid concentration = 1% (v/V)] were mixed at the ratio shown in Table 2 to prepare a chitosan-athelocollagen mixed solution. Polystyrene petri dish (Methylcon 3001)
(diameter = 35fi, depth: 10m). After naturally drying this polystyrene petri dish and forming a thin film of chitosan-atelocollagen complex on the bottom surface, the thin film of chitosan-atelocollagen complex was
Alternate layers of Oc1n were placed, and the chitosan-athelocollagen complex was cross-linked by irradiation with a 30 W ultraviolet lamp, and the boristyrene Petri dish was left in an ammonia atmosphere for 20 minutes.

This polystyrene petri dish was washed twice with M distilled water, and once with Dulbets choline buffer, and the chitosan-
A polystyrene Petri dish with a thin film of atelocollagen interstitial complex formed on the bottom was prepared.

Table 2: M@ ratio of chitosan and atelocollagen in melt-forged chitosan-atelocollagen mixture (R test wear test) Instead of the polymer-coated Petri dish in Example 1, 8M of the chitosan-atelocollagen Wll complex was formed on the bottom surface. A #111 cell adhesion test was conducted in the same manner as in Example 1 using a polystyrene petri dish.

The results were as shown in Figure 7.

In Figure 7, (-0-3 is sample B8-A (chitosan/atelocollagen = 30/70), C-・-)
Trial 8g-8 (Chitosan/Atelocollagen: 20
/80), (-Δ-) is sample 'B8-C (chitosan/athelocollagen: = lO/90), (-low) is sample 8-DC atelocollagen-100), and (-
) is an untreated polystyrene Petri dish (Methylcon 3).
001) This is the congratulations of the chick embryo m vertebral follicle in (Tama).

(Cell Proliferation Test) Instead of the polymer-coated petri dish in Example 1, the chitosan-athelocollagen zm? 1 [A cell proliferation test was conducted in the same manner as in Example 1 using a polystyrene petri dish with a thin film of merging formed on the bottom surface.

The results were as shown in Figure 8.

In Figure 8, (-〇-), (-・-), (-△-)
, (-Rho) and (-Layer 1) are the N increase rates of chicken pyramidal cells, as in FIG.

(Discussion) According to Figure 7, the chitosan-atheloflagen complex (chitosan/athelocollagen = 30/70.2
0/80 and 10/90) are found to adhere chicken fibroblasts to atelocollagen and untreated polystyrene Petri dishes better.

According to FIG. 8, the chitosan-atelocollagen chestnut 1 degree combination (chitosan/atelocollagen = 20/80 and 10/90) proliferated chick embryo 1111 tissue in the same way as atelocollagen and untreated polystyrene petri dishes; In addition, chitosan-atelocollagen cross-linked complex (chitosan/atelocollagen =: 30
/70).

[yI! Effects of IBA]

Of cells? Since it exhibits good stage I attachment and cell proliferation, and has sufficient mechanical strength, it can be used for large-ffi culture of animal cells.

Further, since it is easily precipitated by adding a solution of a basic substance, its production is easy.

[Brief explanation of the drawing]

Figure 1 shows the cells of Example 1! FIG. 2 is a chart showing the results of the cell proliferation test of Example 1. FIG. 3 is a chart showing the results of the cell IMIi test of Example 2. FIG. 4 is a chart showing the results of the cell proliferation test of Example 2. A chart showing the results of the cell increase N test, FIG. 5 is a chart showing the results of the cell adhesion test of Example 3,
FIG. 6 is a chart showing the results of the cell proliferation test of Example 3, FIG. 7 is a chart showing the results of the cell adhesion test of Example 8, and FIG. 8 is a chart showing the results of the cell proliferation test of Example N8. This is a chart showing. Applicant Nippon Biochemicals Co., Ltd. Agent Patent Attorney Tsu 1) Numa 1 Figure 1 Mio Figure 2

Claims (1)

[Scope of Claims] (1) A cell culture substrate characterized by containing a chitosan-collagen complex as an active ingredient. (2) Adding a basic solution to an acidic solution of chitosan in the presence of collagen,
1. A method for producing a cell culture substrate of a chitosan-collagen complex, which comprises precipitating chitosan to form a chitosan-collagen complex. (3) A basic solution is added to an acidic collagen solution in the presence of chitosan to precipitate collagen, and chitosan-
A method for producing a substrate for cell culture of a chitosan-collagen complex, characterized by forming a collagen complex.