JPH08317786A - Cell culture base modified with saccharide chain polymer and cell culture using the same - Google Patents

Abstract

PURPOSE: To culture base capable of controlling change in form, proliferation and function in cell culture based on a specific interaction between a saccharide chain polymer and a cell and to provide a method for cell culture using the cell culture base. CONSTITUTION: This cell culture device is obtained by modifying the surface of a base such as a laboratory dish with at least one or preferably more saccharide chain polymers. Culture is carried out on the cell culture base to control change in form, proliferation and function. The cell culture base is readily produced, has high function and is inexpensive. Both the cell culture base and a method for cell culture can control not only cell adhesiveness but also change in form, proliferation and function of cell.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a cell culture substrate, and in particular, the surface of the substrate is modified with at least one sugar chain polymer so that the morphological change, proliferation and function of cells can be controlled. The present invention relates to a cell culture substrate and a cell culture method using the same.

[0002]

2. Description of the Related Art Recent advances in sugar chain engineering have been remarkable. For example, as a biopolymer having a sugar chain,
Proteoglycans of the cell wall of plant cells that contribute to cell stabilization, glycolipids that affect cell differentiation, proliferation, adhesion, migration, etc., and glycoproteins involved in cell-cell interaction and cell recognition, etc. However, the mechanism by which sugar chains of these macromolecules control high-precision biological reactions while acting, assisting, amplifying, regulating, or inhibiting functions of each other is gradually being clarified.

Furthermore, if the relationship between such sugar chains and cell differentiation / proliferation, cell adhesion, immunity, and cell carcinogenesis is clarified, this sugar chain engineering and medical, cell engineering, or organ engineering is performed. It can be expected that new developments will be made by closely linking and.

[0004] As one example, sugar chains on the cell surface and sugar chains
-Research on the role of sugar chains in the occurrence of diseases caused by abnormal interactions between receptors or in the infection of viruses such as AIDS is becoming active. In addition, studies on the involvement of sugar chains in cell-cell interactions and cell-matrix interactions have also become important for understanding biological reactions.

The inventors of the present invention have been conducting intensive research focusing on the specific affinity of sugar chains. For example, galactose is used as a model of a ligand for an asialoglycoprotein (abbreviated as ASGP) receptor. (N-p-vinylbenzyl- [O-β
-D-galactopyranosyl- (1 → 4) -D-gluconamide]) (common name: polyvinylbenzyl lactone amide, hereinafter abbreviated as PV-LA) was designed and synthesized.

[0006] In general, the surface of a cell culture vessel such as a petri dish is coated with an adhesive protein such as collagen, fibronectin, laminin, etc. to enhance the adhesiveness of cells. Mooney et al.
DN is decreased when A synthesis is reduced and differentiation function is promoted, and when adhesion proteins are coated at low density and cells are grown at low density.
It has been reported that A synthesis is increased and the differentiation function is suppressed (Mooney, et al., J. Cell. Physiol. 151 , 4
97-505 (1992)). That is, it has been suggested that the cell adhesion form and its differentiation function are correlated to some extent.

[0007] The present inventors have used the above-mentioned petri dish coated with PV-LA to selectively select hepatocytes through the specific affinity between PV-LA and the asialoglycoprotein receptor on the surface of hepatocytes. It was found that the three-dimensional self-assembly was guided by the fact that it was adhered to, and the adhesive form was characteristic. These phenomena are not observed in a petri dish coated with a conventional adhesive protein such as collagen or fibronectin (Yoshifumi Yura, Toshihiro Akaike, "Cell Culture", Volume 19, 317-32).
2 1993).

Furthermore, the present inventors have synthesized many sugar chain polymers and extensively studied cell culture on a cell culture substrate modified with these sugar chain polymers. The inventors have found that the degree of change in the adhesive morphology varies depending on the type of polymer, and have completed the present invention. By modifying the surface of the base material with a sugar chain polymer, not only cell adhesion but also
No attempt has yet been made to control its morphological change, proliferation, and function.

[0009]

That is, the object of the present invention is to utilize the morphological change of cells based on the specific interaction between sugar chain macromolecules and cells, and to improve the morphology, proliferability and function in cell culture. The purpose is to provide a cell culture substrate that can be controlled and establish a cell culture method using the same.

[0010]

This problem can be solved by a cell culture substrate characterized in that the surface of the substrate is modified with at least one sugar chain polymer.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION The cell culture substrate of the present invention will be described in detail below. The cell culture substrate of the present invention includes a petri dish,
Any of the substrates conventionally used for cell culture, such as flasks, plates, cuvettes, films, fibers, beads, etc., or any other substrate that can be used for cell culture can be used. These substrates are glass,
It may be made of any inorganic material such as quartz or an organic material such as polystyrene, polypropylene or polyethylene, but is preferably made of a material having heat resistance and water resistance such that it can be sterilized.

[0012] Particularly, the synthetic polymer material is preferable in terms of cost and moldability, and is preferably hydrophobic because of sugar chain polymer adsorption. For example, when a sugar chain polymer having polystyrene or a derivative thereof as a main chain is used, it is preferable to use a base material having polystyrene or a derivative thereof as a main raw material.

The cell culture substrate of the present invention comprises the above substrate surface modified with a sugar chain polymer. The sugar chain polymer used in the present invention is composed of a main chain composed of a synthetic polymer and a side chain composed of a sugar chain.

The synthetic polymer is not particularly limited as long as it can be introduced with a sugar chain as a side chain and can be polymerized by a usual method. For example, polystyrene, vinyl chloride, polyethylene, polyethylene terephthalate, polypropylene, or those thereof. It is preferable to use a vinyl polymer such as a derivative. In particular, it is preferable to use a synthetic polymer composed of polystyrene or a derivative thereof which is widely used as a material for petri dishes because the adsorptivity to the surfaces of petri dishes and the like is improved.

Examples of sugar chains forming the sugar chain polymer include glucose, galactose, lactose, mannose, N-acetylglucosamine, laminaribiose, uronic acid-related substances, monosaccharides such as sulfated sugars, oligosaccharides and the like. Any of them can be used, and it is not particularly limited as long as it can retain a sugar residue that specifically interacts with cells in a state of being bound as a side chain to the synthetic polymer.

In these sugar chain polymers, it is preferable that the synthetic polymer and the sugar chain are bound to each other by a covalent bond such as an amide bond or an ester bond. For example, it is preferable to form an amide bond between the amino group of p-aminomethylstyrene and the terminal carbonyl group of the lactonized sugar. In addition, the synthetic polymer and the sugar chain may be polymerized by binding the monomer forming the synthetic polymer and the sugar chain molecule,
A sugar chain may be bound after polymerizing a monomer having a functional group capable of binding. However, since the number of sugar chains introduced into one molecule can be controlled, it is preferable to polymerize a monomer having a sugar chain bonded thereto.

The sugar chain polymer is preferably a homopolymer obtained by homopolymerizing a sugar chain-bonded monomer, but may be a copolymer with another monomer.
The molecular weight of the sugar chain polymer is not particularly limited as long as it forms micelles and is solubilized when the sugar chain polymer is dissolved in an aqueous solution. In this case, those of about 30,000 to 50,000 are usually used.
However, for example, in the case of a copolymer with a monomer having a hydrophilic group, there is a hydrophilic group that maintains water solubility even if it has a molecular weight outside the above range.
Such sugar chain polymers are also included in the scope of the present invention.

In the cell culture substrate of the present invention, among the above sugar chain polymers, for example, the following formulas (1) to (10)
A sugar chain polymer having a polystyrene derivative as a main chain represented by is particularly preferably used.

[0019]

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[0020]

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[0021]

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[0022]

[Chemical 4]

[0023]

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[0024]

[Chemical 6]

[0025]

[Chemical 7]

[0026]

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[0027]

[Chemical 9]

[0028]

[Chemical 10]

Poly (Np-vinylbenzyl- [O-β-D-galactopyranosyl- (1 →
4) -D-Gluconamide]) (hereinafter abbreviated as PV-LA) is obtained by homopolymerizing a monomer synthesized from p-aminomethylstyrene and lactose,
It has a β-galactose residue.

Poly (N-p-vinylbenzyl- [O-α-D-glucopyranosyl- (1 → 4)-] represented by the above formula (2)
D-Gluconamide]) (hereinafter abbreviated as PV-MA)
Is obtained by homopolymerizing a monomer synthesized from p-aminomethylstyrene and maltose, and has a glucose residue.

Poly (Np-vinylbenzyl- [O-β-D-mannopyranosyl- (1 → 4)-] represented by the above formula (3)
D-mannamide]) (hereinafter abbreviated as PV-Man)
Is obtained by homopolymerizing a monomer synthesized from p-aminomethylstyrene and mannobiose, and has a mannose residue.

Poly (Np-vinylbenzyl- [O-α-D-galactopyranosyl- (1 →
6) -D-Gluconamide]) (hereinafter abbreviated as PV-MeA) is synthesized from p-aminomethylstyrene and O-α-D-galactopyranosyl- (1 → 6) -D-glucose. It is obtained by homopolymerizing the obtained monomer and has a galactose residue.

Poly (N-p-vinylbenzyl- [O-6-carboxymethyl-β-D-galactopyranosyl- (1 → 4) -OD-6-represented by the above formula (5) Carboxymethyl-gluconamide]) (hereinafter abbreviated as PV-LACOOH) is PV-LA obtained by homopolymerizing a monomer synthesized from p-aminomethylstyrene and lactose.
Is carboxymethylated and has a carboxymethylated galactose residue.

Poly (3-O-4 'represented by the above formula (6)
-Vinylbenzyl-D-glucose) (hereinafter abbreviated as PV-G) is obtained by homopolymerizing a monomer synthesized from p-chloromethylstyrene and glucose, and has a glucose residue.

Poly (N-p-vinylbenzyl- [O-2-acetamido-2-deoxy-β-D-represented by the above formula (7)
Glucopyranosyl- (1 → 4) -OD-2-acetamido-
2-deoxy-β-D-glucopyranosyl- (1 → 4) -O-
D-2-acetamido-2-deoxy-β-D-gluconamide]), poly (Np-vinylbenzyl- [O-2-acetamido-2-deoxy-β-represented by the above formula (8). D-glucopyranosyl- (1 → 4) -O-D-2-acetamido-2-
Deoxy-β-D-gluconamide]) or a mixture thereof (hereinafter abbreviated as PV-GlcNac) both have N-acetylglucosamine residues.

The poly (N-p-vinylbenzyl-D-gluconamide) represented by the above formula (9) (hereinafter abbreviated as PV-GA) is prepared by ring-opening D-glucose to form p-aminomethyl. It is obtained by homopolymerizing a monomer bound to styrene.

Poly (Np-vinylbenzyl- [O-β-D-glucopyranosyl- (1 →
3) -D-Gluconamide]) (hereinafter abbreviated as PV-Lam) is obtained by homopolymerizing a monomer synthesized from p-aminomethylstyrene and laminaribiose, and β1 → 3 glucose Have residues.

As a method for modifying the base material with the sugar chain polymer in the cell culture base material of the present invention, a physicochemical method such as adsorption or a chemical method such as covalent bonding can be applied. Is preferred. In the case of modifying by adsorbing a sugar chain polymer, it can be easily modified by applying a sugar chain polymer solution to the surface of a base material such as a petri dish and distilling off the solvent.

More specifically, for example, a sugar chain polymer solution obtained by using distilled water as a solvent is filtered and sterilized, and then injected into a petri dish or the like, and the sugar chain polymer is modified by allowing it to stand overnight. A cell culture substrate is obtained. Preferably, it is used after being washed several times with distilled water and replaced with a balanced salt solution or the like. Water is preferably used as a solvent for dissolving the sugar chain polymer, but depending on the sugar chain polymer to be used, an organic solvent such as ethanol or methanol or a mixed solvent thereof with water can also be used.

The concentration of the above-mentioned sugar chain polymer solution can be appropriately selected depending on the adsorption amount of the sugar chain polymer, which is set according to the purpose. Usually, the concentration range is from 0.01 to 1000 μg / ml, and preferably. Is 0.1 to 500 μg /
It is about ml, and more preferably about 100 μm / ml. Further, this sugar chain polymer solution may be adjusted each time, but if the stock solution stored is used, the labor can be further saved. In that case,
Since sugar chain macromolecules in the stock solution may be associated with each other, it is preferable to perform appropriate ultrasonic treatment before use.

In the cell culture substrate of the present invention, at least one sugar chain polymer is used to modify the surface of the base material, but it is preferable to use two or more kinds of sugar chain polymers in combination. Each cell has a unique sugar chain recognizing property, and the present inventors have found that the degree of morphological change of cells on the cell culture substrate varies depending on the type of sugar chain to be modified. Therefore, by using a cell culture substrate modified by combining two or more kinds of sugar chain macromolecules having different morphological changes, proliferative properties, and degrees of function, only one sugar chain macromolecule is modified. It becomes possible to express morphological changes, proliferative properties, and functions not found in cell culture substrates.

In the case of modifying by combining two or more kinds of sugar chain macromolecules, the solutions of the respective sugar chain macromolecules may be mixed and the mixed solution may be applied as described above for modification. Also,
After modification with one sugar chain polymer solution, the unmodified portion may be modified with another sugar chain polymer solution. The mixing ratio of these sugar chain polymers can be appropriately selected according to the morphology of the target cell.

Further, in the cell culture substrate of the present invention, it is preferable to modify the entire surface of the substrate for cell culture with a sugar chain polymer, but the invention is not limited thereto, and the modified portion and unmodified And a part may be provided. For example, when sugar chain macromolecules are combined with conventional adhesion proteins such as collagen, synthetic macromolecules such as polystyrene, or natural macromolecules, etc., and modified, a part that specifically interacts with cells and a non-specific part are obtained. It is possible to obtain a cell culture substrate in which

Next, the cell culture method using the cell culture substrate of the present invention will be described with reference to specific examples. For example, when using PV-LA as a sugar chain polymer and culturing hepatocytes on a Petri dish modified with PV-LA, first, hepatocytes are isolated by a known method. The isolated hepatocytes are, for example, William's medium E (William's) containing 5% fetal bovine serum.
sMedium E: abbreviated as WE) and the like, and the cells may be seeded on a PV-LA-modified petri dish to a predetermined cell concentration and incubated.

Although one example of the cell culture method of the present invention has been specifically described above, the present invention is not limited to this.
The cell culture method of the present application is characterized by controlling the morphological changes, proliferative properties, and functions of the cells by culturing on a cell culture substrate modified with a sugar chain polymer. The method itself can be used as it is.

[0046]

【Example】

(Example 1) In the production of the cell culture substrate of the present invention, the concentration of the sugar chain polymer solution used when the sugar chain polymer is adsorbed to modify the substrate surface and the sugar chain polymer solution adsorbed on the substrate surface The relationship with the amount of sugar chain polymer was evaluated. 0.01 to 1000μ
PV-L fluorescently labeled with FITC in the concentration range of g / ml
The aqueous solutions A were prepared, and 1 ml of each of the aqueous solutions was poured into a polystyrene cuvette. After leaving it for one day and night, it was washed 2-3 times with distilled water.

In each of the above PV-LA modified cuvettes,
A nonionic detergent (Tween 20) was injected and sonicated, and the amount of desorbed fluorescent labeled PV-LA was measured using a fluorescence spectrophotometer. The results are shown in FIG. 1 as a graph in which the horizontal axis represents the solution concentration and the vertical axis represents the adsorption amount per unit area. It was confirmed that the two are well correlated and that the adsorption amount of the sugar chain polymer can be adjusted by changing the concentration of the applied solution.

(Example 2) (1) Production of sugar chain polymer modified petri dish A commercially available polystyrene petri dish (Falcon 100) was used as a base material.
8, Becton Dickinson & Company). PV-LA is selected as the sugar chain polymer, and the purified PV-LA is fractionated by ultrafiltration into ones having a molecular weight of about 30,000 to 50,000 and those having a molecular weight of 50,000 or more, and 100 μg of the fraction of about 30,000 to 50,000. It was dissolved in distilled water so that the concentration became / ml and left for 2 hours. Then, 1 ml of this PV-LA aqueous solution was poured into the above-mentioned petri dish, allowed to stand for one day and night, washed with distilled water several times, and replaced with a balanced salt solution to obtain a sugar chain polymer-modified petri dish.

(2) Culture of hepatocytes on petri dish modified with sugar chain polymer Hepatocytes isolated by a well-known method were diluted to 3 × 10 ⁴ cells / ml with WE containing 5% fetal bovine serum, The above-described sugar chain polymer-modified petri dish was seeded so as to have 45 × 10 ⁴ petri dishes and incubated. For comparison, the same culture was carried out on an uncoated polystyrene dish and a polystyrene dish coated with collagen.

After the incubation, 1.5 ml of suspension was collected from each dish and immediately rinsed with balanced salt solution. The number of non-adherent cells in the collected solution was counted with a Coulter counter, and the amount of protein in adherent cells was measured using a coloring reagent. The cell adhesion rate in each dish obtained from these results is summarized in FIG. On the collagen-coated petri dish, both hepatic parenchymal cells and hepatic non-parenchymal cells,
It showed higher adhesion than that on an uncoated polystyrene dish, but was non-selective. However, on the sugar chain polymer-modified petri dish, the adhesion of non-parenchymal cells of the liver was significantly suppressed,
A high selectivity was observed in which only hepatocytes were efficiently adhered.

Further, when the adhesion morphology of those hepatocytes was observed, in the collagen-coated petri dish, flattening of the cells was observed from the early stage of adhesion, and after 48 hours, the flattened cells were cobblestone-shaped over the entire petri dish. I suffered. However, the sugar chain polymer-coated petri dish maintained an almost perfect spherical adhesion form, and after 48 hours, they formed a three-dimensional multicellular aggregate. Further, in this self-assembled hepatocyte, the albumin synthesizing ability was stably maintained as compared with the flattened hepatocyte on the collagen-coated petri dish, and self-aggregation resulted in appropriate differentiation of hepatocyte. It was suggested that was induced.

(Example 3) PV-L as a sugar chain polymer
A, PV-MA, PV-Man, and PV-GlcNac
Was selected, and 4 types of sugar chain polymer-coated petri dishes were produced in the same manner as in Example 2. 3T3 cells were cultured on those polymer-coated petri dishes, and the morphology of the cells after 24 hours was observed with a microscope. The results are shown in FIGS. 3 to 6, respectively. For comparison, similar culturing was performed on an unmodified polystyrene dish and a polystyrene dish coated with collagen to observe the cell morphology. The results are shown in FIGS. 7 and 8, respectively.

On the uncoated and collagen-coated petri dishes, flattened cells were covered on the entire surface, whereas PV
-On Man-modified petri dishes, spherical cells form self-assemblies. Meanwhile, PV-LA, PV-MA, and P
Although flattening of cells occurs on the V-GlcNac-modified petri dish, the flattened cells tend to form a three-dimensional aggregate, and the degree thereof varies depending on the type of sugar chain.

Example 4 PV-L as sugar chain polymer
A, PV-MA, PV-Man, and PV-GlcNac
Was selected, and 4 types of sugar chain polymer-coated petri dishes were produced in the same manner as in Example 2. Human cancer cells (WRL) were cultured on those polymer-coated petri dishes, and the morphology of the cells after 24 hours was observed with a microscope. The results are shown in FIGS. 9 to 12, respectively. For comparison, similar culturing was performed on an unmodified polystyrene dish and a polystyrene dish coated with collagen to observe the cell morphology. The result is shown in Figure 1.
3 and 14 respectively.

On the uncoated and collagen-coated petri dishes, the cell growth did not proceed so much, whereas on the sugar chain polymer-modified petri dishes, PV-LA, PV-MA, P
Proliferation of cells is progressing in the order of V-GlcNac-modified petri dish, and 3 cells are present on the PV-Man-modified petri dish.
It is dimensionally aggregated. In addition, this three-dimensional transformation of cells is achieved by controlling the number of cells to be entrained in PV.
-Can also be generated on LA, PV-MA.

As described above, by culturing cells on the cell culture substrate of the present invention, morphological changes of the cells which are not seen in the uncoated substrate or the collagen-coated substrate occur, and the morphological changes It was revealed that the degree was specific depending on the cell type and the sugar chain polymer type. Therefore,
With a cell culture substrate modified by combining two or more types of sugar chain polymers, it becomes possible to cause a morphological change that cannot be seen with one type of sugar chain polymer, and change the combination and mixing ratio of sugar chains. Thus, the morphological change of cells can be controlled.

Example 5 PV-L as a sugar chain polymer
A, PV-MA, PV-Man, and PV-GlcNac
Was selected, and four types of sugar chain polymer-coated petri dishes were produced in the same manner as in Example 2. 3T3 cells adjusted to 1.5 × 10 ⁵ cells / ml were seeded on the sugar chain polymer-coated petri dish, and the proliferation of the cells was traced every 3 hours from the 9th hour after the start of culture. The results are shown in Fig. 15. The vertical axis in the figure shows the percentage of cells that are taken from the PI uptake to S phase. For comparison, perform the same culture on untreated polystyrene dish and petri dish coated with collagen,
The proliferation of cells was observed. This result is also shown in FIG.

Under the above conditions, 3T3 cells were
Three-dimensionally aggregated on LA and PV-MA. On the other hand, on other sugar chain polymers, collagen, and polystyrene,
The cells spread and proliferated. Three-dimensional PV-LA and P
It was revealed that the cells on V-MA did not enter the growth phase within 24 hours of culture. Polystyrene, PV-M
It was found that about 40% of the cells entered the proliferative phase and started to proliferate within about 15 hours after culturing with an and PV-GlcNac, and after about 16 hours on collagen.

As described above, by culturing the cells on the cell culture substrate of the present invention, it becomes possible to control the proliferation of the cells, which is not found in the uncoated substrate or the collagen-coated substrate. It was clarified that the degree is specific depending on the number of cells entrained and the type of sugar chain polymer.

Example 6 PV-Lam, which is one of the sugar chain polymers used in the present invention, was synthesized as follows. (1) Lactarimination of laminaribiose 10 g of laminaribiose was dissolved in 5 ml of distilled water, and the solution was diluted with 45 ml of methanol. The diluted solution was added to a methanol solution of iodine (17.1 g / 200 ml) heated at 40 ° C., left for 30 minutes, and then a 4% potassium hydroxide / methanol solution was added until the color of iodine disappeared. It was dripped slowly. The color of the element disappeared with the addition of 400 ml of potassium hydroxide / methanol solution. Subsequently, the reaction solution was ice-cooled, and the deposited precipitate was collected by filtration. The precipitate was washed with cold ethanol and cold ether in this order, and recrystallized from ethanol / water (9: 1 (weight / weight)) to obtain a potassium salt.

The obtained potassium salt was allowed to stand overnight at 4 ° C., and then ion-exchange resin (Amberlite IR-12B) was used.
(H ⁺ type) and ion-exchanged, followed by freeze-drying to obtain an acid type (laminaribionic acid). Methanol was added to the acid fraction and concentrated under reduced pressure to give crystals. The operation of adding a small amount of methanol to the crystals to dissolve them, further adding ethanol and dehydrating and concentrating was repeated 5 times, and then dried under reduced pressure to obtain about 7 g of laminaribiolactone.

(2) Coupling with p-aminomethylstyrene 7 g of the above laminaribiose lactone was added to 50 parts of methanol.
It was dissolved in ml, and a solution of p-aminomethylstyrene in methanol (2.5 g / 0.5 ml) was added to the solution under heating under reflux. After heating under reflux for 120 minutes, acetone 2
It was crystallized by adding 00 ml. The obtained crystals were recrystallized twice from methanol to give 3.5 g (yield 39%).
P-vinylbenzyl laminaribiose lactone amide (formal name: Np-vinylbenzyl- [O-β-D-glucopyranosyl- (1 → 3) -D-gluconamide], hereinafter VB-
(Abbreviated as Lam) was obtained.

(3) Structure confirmation of VB-Lam The structure of the obtained V-Lam was analyzed by ¹ H-NMR and ¹³ C-NM.
Confirmed by R. ^{In the 1} H-NMR spectrum, 3.2
A peak derived from laminaribiose at ~ 4.0 ppm,
A peak derived from a vinyl group at 5.2 to 6.8 ppm,
A peak derived from a benzene ring was found in the styrene skeleton at around 7.4 ppm. ^{In the 13} C-NMR spectrum, 4
Peak of NH-CH ₂ in 3.439ppm, 62.27
Laminaribiose peak at 7-78.198 ppm,
Vinyl group double bond peak at 101.018 ppm,
A peak of benzene having a styrene skeleton was observed around 127.415 to 138.502 ppm, and a peak of C = O of an amide bond was observed at 175.500 ppm, confirming the structure of VB-Lam.

(4) Polymerization VB-Lam (2 g) synthesized above and having a confirmed structure was dissolved in DMSO (2 ml), and AIB was used as a polymerization initiator.
N (1 mg) was added. The solution was heated to 60 ° C. under nitrogen and polymerized for 24 hours to obtain PV-Lam (about 2 g). The number average molecular weight of this PV-Lam is about 40,000.
It was 0. A sugar chain polymer having a β1 → 3 glucose structure such as PV-Lam has not been known so far.

[0065]

EFFECTS OF THE INVENTION The cell culture substrate of the present invention can be easily produced by simply modifying the surface of a conventionally used substrate with a sugar chain polymer. Particularly, by using a sugar chain polymer having polystyrene or a derivative thereof as a main chain, it is possible to easily modify a base material having polystyrene as a main raw material, and thus such a cell culture base material is highly functional and inexpensive and is disposable. It can also be a product.

The cell culture substrate and cell culture method of the present invention are:
It is based on the specific interaction between cells and sugar chains, and can control not only cell adhesion but also cell morphological changes, proliferative properties, and functions. Therefore, it becomes possible to obtain cells of any morphology, and it is possible to apply them to, for example, a hybrid artificial organ.

[Brief description of drawings]

FIG. 1 is a graph showing a relationship between a PV-LA aqueous solution concentration and a PV-LA adsorption amount.

FIG. 2 is a graph showing the specificity of hepatocyte adhesion on an unmodified polystyrene petri dish, a petri dish modified with collagen and PV-LA.

FIG. 3 3T3 cultured on a PV-LA-modified petri dish
It is a photograph showing the morphology of cells.

FIG. 4 3T3 cultured on a PV-MA modified petri dish
It is a photograph showing the morphology of cells.

FIG. 5: 3T cultured on a PV-Man modified petri dish
It is a photograph showing the morphology of 3 cells.

FIG. 6 is a photograph showing the morphology of 3T3 cells cultured on a PV-GlcNAc-modified petri dish.

FIG. 7 is a photograph showing the morphology of 3T3 cells cultured on an uncoated polystyrene dish.

FIG. 8: 3 cultured on a petri dish coated with collagen
It is a photograph showing the morphology of T3 cells.

FIG. 9: WRL cultured on a PV-LA-modified petri dish
It is a photograph showing the morphology of cells.

FIG. 10: WR cultured on a PV-MA modified petri dish
It is a photograph showing the morphology of L cells.

FIG. 11: W cultured on a PV-Man modified petri dish
It is a photograph showing the morphology of RL cells.

FIG. 12 is a photograph showing the morphology of WRL cells cultured on a PV-GlcNAc-modified petri dish.

FIG. 13 is a photograph showing the morphology of WRL cells cultured on an uncoated polystyrene dish.

FIG. 14 is a photograph showing the morphology of WRL cells cultured on a collagen-coated dish.

FIG. 15 is a graph showing the results of controlling the growth of 3T3 cells using a sugar chain polymer-modified petri dish.

Claims (7)

[Claims] 1. A cell culture substrate obtained by modifying the surface of a substrate with at least one sugar chain polymer. 2. The cell culture substrate according to claim 1, wherein the substrate surface is modified with two or more kinds of sugar chain polymers. 3. The cell culture substrate according to claim 1 or 2, wherein the substrate is selected from a petri dish, a flask, a plate, a cuvette, a film, a fiber and beads. 4. The cell culture substrate according to claim 1, wherein the substrate is mainly composed of polystyrene and its derivative. 5. The sugar chain polymer is poly (Np-vinylbenzyl- [O-β-D-galactopyranosyl- (1 → 4) -D-gluconamide]), poly (N- p-vinylbenzyl- [O-α-D-glucopyranosyl- (1 → 4) -D-gluconamide]), poly (Np-vinylbenzyl- [O-β-D-mannopyranosyl- (1 → 4)) -D-mannanamide]), poly (Np-vinylbenzyl- [O-α-D-galactopyranosyl- (1 → 6) -D-gluconamide]], poly (Np-vinylbenzyl-) [O-6-carboxymethyl-β-D-galactopyranosyl- (1 → 4) -OD-6-carboxymethyl-gluconamide]), poly (3-O-4′-vinylbenzyl-D -Glucose), poly (Np-vinylbenzyl- [O-2-acetamide-2
-Deoxy-β-D-glucopyranosyl- (1 → 4) -OD-
2-acetamido-2-deoxy-β-D-glucopyranosyl
-(1 → 4) -OD-2-acetamido-2-deoxy-β-
D-Gluconamide]) and / or poly (Np-vinylbenzyl- [O-2-acetamido-2-deoxy-β-D-
Glucopyranosyl- (1 → 4) -OD-2-acetamido-
2-deoxy-β-D-gluconamide]), poly (Np-vinylbenzyl-D-gluconamide), and poly (Np-vinylbenzyl- [O-β-D-glucopyranosyl- (1 → 3) -D-Gluconamide]), the cell culture substrate according to claim 4. 6. A cell culture method, which comprises culturing cells on the cell culture substrate according to any one of claims 1 to 5 and controlling morphological changes, proliferative properties, and functions of the cells. 7. The morphological change, proliferative property, and function of cells are controlled by using two or more kinds of sugar chain polymers and changing the mixing ratio of these sugar chain polymers. Cell culture method.