JP2654744B2 - Culture substrate - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel culture substrate used for culturing cells in vitro, and more particularly, to a method for stably maintaining and growing nerve cells in vitro. The present invention relates to a novel culture substrate that can be used, and a method for stably culturing nerve cells using the culture substrate.

[0002]

2. Description of the Related Art At present, great attention has been paid to the nervous system of living organisms due to the progress of cell biology and the promotion of advanced medical treatment, and research from various angles has been advanced. However, to date, neuronal cells can be propagated in vitro only in the early stages of development, and cannot be proliferated in ordinary systems. More importantly, expressing the same functions in vivo as in vivo remains as a major challenge.

[0003] Therefore, various attempts have heretofore been made to develop a culture substrate having a high level of function useful for use in such research, and many techniques for culturing neurons have been developed. By the way, generally, culture of nerve cells
Typically, a method of coating a glass or plastic substrate with a cell adhesive protein, collagen, laminin, fibronectin, or the like, or using a method of coating a basic polyamino acid, polylysine, polyornithine, or the like is used as a typical example. can give.
By using such a method, the adhesion of nerve cells to a substrate has recently been increased, and stable culture can be performed.

[0004] As described above, various culture media suitable for culturing nerve cells have recently been developed, and the adhesion of nerve cells to a substrate has been increased. Has been developed, but at the same time the adhesion of glial cells mixed in this system will increase. It is known that cells of the nervous system include nerve cells responsible for information transmission and glial cells that maintain a microenvironment in which nerve cells exist. These glial cells, unlike nerve cells, have the property of actively dividing and growing in vitro.

[0005] That is, a useful culture substrate for culturing nerve cells and a culture system such as a culturing method have been developed so far, but glial cells always grow if cultured in the conventional system. Come together. This, of course, hinders the survival of neurons. Therefore, in the usual culture so far, it has been common to take measures to add a growth inhibitor such as cytosine arabinoside and 5-fluorodeoxyuridine to the culture medium. This method is harmful to cells, and it is a negative method for stable culture of nerve cells, and its use is limited. There was a strong demand in the industry to develop new technologies that could be eliminated.

[0006]

Therefore, the present inventor has proposed:
Based on this situation, as a result of conducting various studies to establish a new technology capable of stably culturing nerve cells without using such a harmful growth inhibitor,
The present inventors have found that the characteristics of the substrate to be coated vary greatly depending on the substrate to be coated, and have further studied to complete the present invention.

That is, the object of the present invention is to
It is an object of the present invention to provide a culture substrate capable of suppressing the proliferation of glial cells in vitro and stably culturing nerve cells.

Another object of the present invention is to provide a culture substrate for neural cells capable of suppressing the growth of glial cells without adding a growth inhibitor used in ordinary culture to a medium. Is to provide.

It is another object of the present invention to provide a method for stably culturing nerve cells using such a culture substrate.

[0010]

The present invention for achieving the above object is constituted by the following technical means (1) to (3). (1) A culture substrate obtained by coating polyhydroxyethyl methacrylate with a basic polymer.

(2) The culture substrate according to the above (1), wherein the basic polymer is selected from polylysine, polyornithine, polyethyleneimine, polyacrylamine and polyalginic acid.

(3) A method for stably culturing nerve cells in vitro, comprising using the culture substrate according to (1).

Next, the present invention will be described in more detail. In the culture substrate of the present invention, polyhydroxyethyl methacrylate, which is a substrate to be coated, is a non-cell-adhesive polymer and can form a substrate with the polymer alone, but also polyhydroxyethyl methacrylate on glass or plastic. By taking a method such as forming a methacrylate layer, the substrate to be coated can be formed in a complex form. In this case, for example, a glass microcarrier (diameter: 90 to 210 μm, manufactured by ICN Biomedicals) or the like used for cell culture is immersed in an ethanol solution of the present polyhydroxyethyl methacrylate to form a composite with a glass microcarrier. A substrate to be coated can be formed. Further, a material cast into a mold having a predetermined shape can be used.

On the other hand, as the basic polymer which is a cell adhesive polymer, various polymers can be used as long as they do not show cytotoxicity. For example, polylysine, polyornithine, polyethyleneimine, polyallylamine, and polyalginic acid are exemplified as suitable examples, but are not limited thereto. The culture substrate of the present invention is produced by converting such a basic polymer into an aqueous solution and coating it on the above-mentioned cell non-adhesive polymer, polyhydroxyethyl methacrylate.

Further, the method for producing the culture substrate of the present invention will be described. When a substrate to be coated with polyhydroxyethyl methacrylate is formed, for example, a 10% solution of polyhydroxyethyl methacrylate in ethanol or a solution equivalent thereto is used. Glass, plastic or a metal such as silver, gold or the like that does not exhibit cytotoxicity equivalent thereto, a metal oxide such as indium-tin oxide, after coating in a layer on a substrate such as a ceramic, It only has to be dried. In this case, the concentration of polyhydroxyethyl methacrylate is not particularly limited, for example,
0.1-12% is exemplified as a suitable thing. Also,
Drying may be performed by a conventional method, and for example, a method of drying in a clean bench under irradiation of an ultraviolet lamp is exemplified as a suitable method.

Next, the coating of the basic polymer is:
For example, the surface of the substrate is covered with a 0.1% basic polymer aqueous solution, left standing at about 4 ° C. for 15 to 20 hours, and then washed with pure water or a substance having the same effect. Although a method of drying in the same manner as described above is exemplified as a suitable method, the present invention is not limited to this, and an appropriate method can be adopted.

Although the form of the culture substrate of the present invention is not particularly limited, for example, those formed in an appropriate form such as a petri dish, a microcarrier, a flask, a bottle, a slide glass, a cover glass, a hollow fiber, and a fiber are suitable. As an example.

The culture substrate of the present invention reliably suppresses the division and proliferation of glial cells, which has conventionally been a problem when culturing nerve cells in vitro, and provides stable survival of neural cells such as nerve cells. The present invention has a property that enables maintenance and growth, and the present invention utilizes a culture substrate having such a property to stably survive and maintain nervous system cells such as nerve cells in vitro. It is possible.

The culture substrate of the present invention thus formed can be used as a substrate for culturing living cells, for example, cells such as adrenal medulla cells. It is useful as a substrate for culturing cells in vitro. Here, the term "neural cells" means not neural cells but neural cells such as nerve central nerve cells and primary peripheral nerve cells.

In order to culture these nerve cells using the culture substrate of the present invention, the nerve cells may be cultured in an appropriate culture solution, for example, in a 37 ° C. carbon dioxide incubator by a conventional method. The culture method itself is not particularly limited. As a culture solution of nerve cells, DME / F-12 was used.
(GIBCO BRL), EME, DME, F-1
2, L15 (GIBCO BRL) and the like are exemplified as suitable ones.

[0021] As described above, the culture substrate of the present invention is obtained by using polyhydroxyethyl methacrylate, a non-cell-adhesive polymer, as a substrate to be coated and complexing a basic polymer, a cell-adhesive polymer, as a coating substrate. By using such a culture substrate, it is possible to reliably suppress the proliferation of glial cells, which has been a problem in the conventional culture method, and to stably culture nerve cells. Such characteristics of the polyhydroxyethyl methacrylate are, to the knowledge of the present inventors, no examples reported so far, and are novel findings found by the present inventors.

[0022]

EXAMPLES Next, the present invention will be described specifically with reference to examples, but the present invention is not limited to these examples. Example 1 0.1 ml of a 10% ethanol solution of polyhydroxyethyl methacrylate was added to a 35 mm diameter glass petri dish rotated 1,000 times per minute, and dried in a clean bench for 30 minutes under irradiation of an ultraviolet lamp. Thereafter, 2 ml of a 0.1% aqueous solution of polyornithine was added and left at 4 ° C. for 18 hours. Then, after washing with pure water and drying,
The culture substrate of the present invention was formed on a glass Petri dish.

The culture solution used for culturing nerve cells is DME
/ F-12 solution (GIBCO BRL) that had been acclimated to primary glial cells was used. The method of acclimation was based on the method of Sean Murphyr et al.
hods in Neurosciences, Vo
l. 2, 33 (1990), Academic Pre
ss company] and cultured primary DM cells
A method of culturing in E / F-12 solution for 2 days was used.

Nerve cells are obtained from rat cerebral brain [Fi
nkbeiner, Steven, and Setev
ens, Charles F: Application
soft Quantitative Measurem
ents for Assessing Glutam
ate Neurotoxicity, Proc. Na
t. Acad. Sci. , (USA), 85, 4071
(1988)], and dispersed and taken out with the enzyme papain to obtain a DME / F-12 solution (GIBCO B
(RL Co., Ltd.) to prepare a suspension. The nerve cells are added to the above culture solution,
N2 additive consisting of insulin, transferrin, sodium selenite, and progesterone (GIBCO
(BRL) was added to the culture solution so as to be diluted 100-fold, and the cells were cultured in a petri dish formed from the culture substrate of the present invention.

Comparative Example 1 For comparison, nerve cells were cultured in the same manner as in Example 1 except that a glass Petri dish coated with polyornithine was used.

In order to compare the culture results of the above example and the comparative example, the culture medium was changed at intervals of 2 to 3 days and cultured for 1 week. In the comparative example, glial cell proliferation was remarkably observed. According to the invention, the proliferation of glial cells is about 1/1 of the comparative example.
It was about 0, the proliferation was remarkably suppressed, and good survival and growth of nerve cells were observed. Thus, it was confirmed that the present invention exerts a unique effect not found in the prior art.

[0027]

As described in detail above, the present invention is a composite of a cell non-adhesive polymer, polyhydroxyethyl methacrylate, as a substrate to be coated and a basic polymer, a cell adhesive polymer, as a coating substrate. The present invention relates to a formed culture substrate, which is suitable for culturing biological cells, particularly nervous cells such as nerve cells, and ensures the proliferation of glial cells, which has been a problem in conventional culture methods. Can be suppressed. The culture substrate according to the present invention is used when culturing living cells in vitro, and can be used regardless of the cell type, but is particularly preferably used for nerve cells.

Claims (3)

(57) [Claims] 1. A method for inhibiting the growth of glial cells in which polyhydroxyethyl methacrylate is coated with a basic polymer.
Culture substrate for central nervous cells . 2. The basic polymer is polylysine, polyornithine, polyethyleneimine, polyacrylamine,
The culture substrate for central nervous cells according to claim 1, which is selected from polyalginic acid. 3. A method for stable culture of central nervous cells in vitro using the culture substrate according to claim 1.