JPH0191770A - Substrate for cell culture - Google Patents

Abstract

PURPOSE:To obtain an easily handleable, low-cost cell culture substrate effective in promoting the growth and proliferation of cells, by bonding a bio-information molecule to a polymer having carboxyl group and amino group by coupling reaction. CONSTITUTION:The objective culture substrate suitable in the field of medical material, e.g., artificial substrate can be produced by coupling (A) a polymer having carboxyl group and/or amino group (e.g., carboxymethyl cellulose or polyaspartic acid) with (B) a bio-information molecule (preferably bovine insulin, bovine epithelocyte growth factor, bovine transferrin, etc.), thereby grafting and immobilizing the component B.

Description

Detailed Description of the Invention [Field of Industrial Application] The present invention relates to a cell culture substrate that can be suitably used in the field of medical materials such as a tissue cell culture substrate and an artificial substrate, and more specifically, The present invention relates to a cell culture substrate based on a bioinformation molecule-fixed polymer, which is inexpensive, easy to handle, and has practical advantages such as being able to obtain a high cell growth rate.

[Conventional technology] Conventionally, as a technique for culturing and propagating cells such as mouse fibroblasts, cells were cultured by adding K in a medium containing various nutrients and biological information molecules such as insulin, which is a cell growth/proliferation factor. Methods of promoting growth are known.

However, such conventional media are expensive and complicated to handle, and have drawbacks such as insufficient cell growth promotion and promotion effects.

An object of the present invention is to provide a polymer-based cell culture substrate that is inexpensive, easy to handle, and has excellent cell growth and proliferation effects, which can be used as a substitute for the conventional culture medium having the above-mentioned problems. With the goal.

[Means for solving the barber point] As a result of intensive research to solve the above problem, the present inventor has developed a method for adding biological information molecules, which are cell growth and proliferation factors, to a polymer having a specific functional group. The present inventors have discovered that an immobilized polymer material that is immobilized by a coupling reaction satisfies the object of the present invention as a cell culture substrate, and has completed the present invention based on this knowledge.

That is, the structure of the present invention is a cell culture substrate characterized in that a biological information molecule is bonded to a polymer having a carboxyl group and/or an amine group by a coupling reaction.

The cell culture substrate of the present invention uses a polymer having a carboxyl group and/or an amino group as a carrier, and a bioinformation molecule is bonded to this polymer by a coupling reaction.

The polymers to which carboxyl groups and/or amino groups are bonded include known polymers having carboxyl groups and/or amine groups, or hydrolysis, hydrogenation, and oxidation reactions on the main chain or side chain of the raw material polymer. Any polymer into which a carboxyl group and/or an amino group has been introduced or generated by various chemical reactions such as reaction with a compound having a carboxyl group and/or an amino group can be used.

Here, the amine group is usually at least 1
Amino groups having N-H bonds are preferred, especially -
NQ2 is preferred.

Examples of the carboxyl group-containing polymer include carboxymethylcellulose Tyrsephadex, Bio-Get CM-100
(trade name), polyaspartic acid, polygalacturonic acid, etc., and can be obtained from various raw material polymers as shown below.

The raw material polymers include, for example, polyesters having an ester bond in the main chain such as polyalkylene terephthalate, polyamides having an amide group in the main chain such as nylon, polyacrylate, polymethacrylate, etc. having an ester bond in the side chain. Polymers with amide bonds in their side chains such as polyacrylamide and polymethacrylamide; polymers that can introduce amine groups into their side chains by hydrogen reduction such as polyacrylonitrile; Spacers with amino groups or carboxyl groups such as cellulose, sepharose, etc. (carboxyl in the polymer side chain), cellulose, sepharose, etc.

Various polymers can be mentioned, such as reactive polymers into which compounds for introducing groups and/or amino groups can be easily introduced.

In addition, these raw material polymers are subjected to hydrolysis as described above,
By suitably performing chemical operations such as reaction with a spacer, it is possible to obtain a polymer in which the content of carboxyl groups and ami-7 groups is adjusted. / Or as a polymer containing an amino group, it can be used, among others, the surface of a polymer having an ester bond such as polyalkylene terephthalate or polymethacrylate is moderately hydrolyzed to introduce carboxyl groups onto the surface of the polymer. Polymers can be suitably used.

This hydrolysis method is not particularly limited, and known methods such as dilute acid hydrolysis, saponification followed by acid treatment, etc. can be used. The amount can be controlled by adjusting the temperature, reaction time or other conditions of this hydrolysis.

Note that the content of carboxyl groups in the polymer can be determined by a conventional method using rhodamine 6G.

Examples of the polyalkylene terephthalate include polyethylene terephthalate and polybutylene terephthalate. Among these, polyethylene terephthalate is particularly suitable.

Examples of the polymethacrylate include polymethyl methacrylate, polyethyl methacrylate, polybutyl methacrylate, poly(2-ethylbutyl methacrylate), polyhexyl methacrylate, polyoctyl methacrylate, polycyclohexyl methacrylate,
Examples include polybenzyl methacrylate and polyphenyl methacrylate.

Among these, polymethyl methacrylate is particularly suitable.

The spacer is a compound that has a reactive group that can be bonded to the raw material polymer through a chemical bond, and also has an amino group and/or a carboxyl group.

Specifically, examples include polyamines such as ε-aminocaproic acid, glycyl-glycyl-glycine, hexamethylene diamine, dodecamethylene diamine, aminocarboxylic acids, polycarboxylic acids, or derivatives thereof, and furthermore, those containing amines at the molecular ends. Examples include polyethylene glycol having a carboxyl group at the molecular end, and polyethylene glycol having a carboxyl group at the molecular end.

These raw material polymers may be used alone, or two
It may be used in combination as a mixture or composition of more than one species, and the polymer having carboxyl group and/or amino group may be used alone.
Two or more types may be used together as a mixture or composition.

The raw material polymer or carbo used.

A polymer having a xyl group and/or an amine 7 group.

There is no particular restriction on the shape of the material, and it may be in any form, such as film, sheet, particulate, powder, fiber, cloth, or block, and may have various shapes depending on the purpose of use. You can choose.

As the biological information molecule, known cell growth and stone factors can be used, but since they are widely used as commercially available products, for example, bovine insulin, bovine epidermal growth factor (EGF), Particularly suitable examples include bovine transferrin and the like.

These biological information molecules may be used alone, or two or more types may be used in combination as long as it does not impede the purpose of the present invention.

The cell culture substrate of the present invention comprises the carboxyl group and/or
Alternatively, it can be obtained by grafting and immobilizing the biological information molecule onto a polymer having 7 amino groups by a coupling reaction.

Immobilization by this coupling reaction is usually carried out by attaching carbodiimide to the carboxyl group and/or amino group in the polymer, particularly on the polymer surface, or to the carboxyl group and/or amine group introduced onto the polymer surface by introducing a spacer. This can be easily carried out by coupling the biological information molecule using a water-soluble carbodiimide (WSC), such as Woodward's reagent, or the like.

The carbodiimide is a compound represented by the following general formula %() (where R and R' represent a monovalent group, such as a hydrocarbon group or a substituted hydrocarbon group). Some of them are commercially available as carbodiimide reagents.

On the other hand, N-ethyl-5-phenylisoxazolium-3'-sulfonate (Woodward reagent K) and the like are known as Woodward reagents.

Among these, water-soluble carbodiimide (WSC
), for example, 1-ethyl-3(3-dimethylaminopropyl)carbodiimide is particularly preferably used.

The coupling reaction conditions for this immobilization are as follows:
It cannot be specified uniformly because it varies depending on the type of bioinformation molecule used, the amount of carboxyl groups and amine groups in the polymer, the type of coupling agent used, etc., but for example, if the carboxyl group in the polymer is In the case of immobilization using , it can usually be carried out by treating in an aqueous solvent with a pH of about 1.0 to 13.0 at a temperature of 0 to 50° C. for about 1 to 24 hours.

There is no particular limit to the amount of this bioinformation molecule immobilized, but the bioinformation molecule is usually 0.01 g g/cm2 or more per unit surface area of the polymer having a carboxyl group and/or an amino group. Preferably 0.1
It is preferable to set it in the range of μg/am2 or more. The amount of immobilization is 0.01. If Bg/as2 is less than 2, when the obtained cell culture substrate is used for cell growth and proliferation, a sufficient growth and proliferation rate of the cells may not be obtained.

The amount of immobilized bioinformation molecules is usually 1
Staining method with sic brilliant blue,
Alternatively, it can be quantified by ATR-FT-IR method.

The cell culture substrate of the present invention obtained in this way is inexpensive compared to conventional culture media, and is based on a polymer substrate, so
It can be used in various shapes as described above depending on the purpose, and is easy to handle.Moreover, when used for promoting the growth and proliferation of cells such as mouse fibroblasts, It has practical advantages such as improved proliferation rate, and can be suitably used, for example, as a tissue cell culture substrate, an artificial substrate, etc. in the field of medical materials.

[Example] Next, the present invention will be specifically explained by showing examples of the present invention.

(Example 1) Bovine insulin-immobilized polyethylene terephthalate and Futakami Gorachiyu■ Polyethylene terephthalate membrane was diluted with 4N NaOH
After hydrolyzing with aqueous solution/CIh0H (volume ratio 1:3) for 30 minutes, 10% citric acid aqueous solution/C:H
30H (volume ratio 1:3), a polyethylene terephthalate film having carboxyl groups was obtained. The amount of carboxyl group is 1.3 x 10-8 mol/c
m2, and absorption based on carboxylade was confirmed by ATR-FT-IR.

■ Under conditions where the pH was adjusted to 7.0 and maintained at 4°C, the polyethylene terephthalate film having carboxyl groups was treated with 1% by weight of water-soluble carbodiimide (WSC).
) in the presence of a bovine insulin solution with a concentration of 30 ILg/m2 for 4 hours to immobilize bovine insulin by grafting.

The amount of bovine insulin immobilized was 0.3 μg/c layer 2, and absorption based on peptide bonds was confirmed by ATR-FT-IH.

Proliferation test of mouse fiber 1. Mouse fibroblast STO 1.06 on the above bovine insulin-immobilized polyethylene terephthalate membrane.
XIO/l! After coating at a concentration of L and culturing for 48 hours while maintaining at 37°C, the number of cells was quantified by the lactate dehydrogenase activity (LDH) method to measure cell growth. It was confirmed that there was no elution of bovine insulin from the immobilized membrane by immersing it in phosphate buffer for 48 hours or more.

The results are shown in Table 1.

(Example 2) The procedure was carried out in exactly the same manner as in Example 1, except that in (1) of Example 1, the immobilization time of bovine insulin was changed to 1 hour.

The results are shown in Table 1.

(Comparative Example 1) In Example 1, a test similar to (2) in Example 1 was conducted using a polyethylene terephthalate membrane on which bovine insulin was not immobilized in place of the polyethylene terephthalate membrane on which bovine insulin was immobilized.

The results are shown in Table 1.

(Comparative Example 2) In Example 1, the test was conducted in the same manner as in Example 1, except that a polyethylene terephthalate membrane on which bovine insulin was adsorbed was used instead of the polyethylene terephthalate membrane on which bovine insulin was standardized at 9J. did.

The results are shown in Table 1.

(Comparative Example 3) In Example 1, Eagle ME was used instead of the polyethylene terephthalate membrane on which bovine insulin was immobilized.
The test was carried out in the same manner as in Example 1, except that a system in which bovine insulin was added to the N medium was used.

The results are shown in Table 1.

Table 1 [Effects of the Invention] The cell culture substrate of the present invention immobilizes biological information molecules such as bovine insulin to the carboxyl group and/or amino group of a specific polymer substrate through a strong chemical bond through a coupling reaction. Since it is a polymer-based cell culture substrate made of a polymeric material, it is cheaper than conventional media, can be used in various shapes depending on the purpose, is convenient to handle, and is highly effective for cell growth and It has a high promoting effect on proliferation, and has practical advantages such as the leakage caused by the use of immobilized bioinformation molecules can be practically ignored and it can be used in a stable state. ing.

That is, according to the present invention, it is possible to provide a new type of cell culture substrate that has the above-mentioned advantages and is extremely superior in practice.

Patent applicant: Idemitsu Kosan Co., Ltd. 1. Do′,′: 2°.

Agent: Patent Attorney Naoki Fukumura”°゛−.

Claims (2)

[Claims] (1) A cell culture substrate characterized in that a biological information molecule is bonded to a polymer having a carboxyl group and/or an amino group by a coupling reaction. (2) The cell culture device according to claim 1, wherein the bioinformation molecule is bovine insulin, bovine epidermal growth factor, or bovine transferrin.