JPH02265477A - Controlling element of growing direction of nerve fiber and production thereof - Google Patents

Abstract

PURPOSE:To stably and readily produce nerve cell in a large amount by providing the surface comprising specific (meth)acrylate resin and having many fine undulations at touching part with nerve cell. CONSTITUTION:A controlling element of growing direction of nerve cell is obtained by notching many fine undulations at touching part with the nerve cell on the surface of a molded element (molding condition: amount of light is 10<3>-10<4>mJ for from several sec to several min/at room temperature, of one or more photo-setting (meth)acrylate resin selected from polyethylene glycol diacrylate expressed by formula I (n is 1-6), epoxy acrylate expressed by formula II, polyethylene glycol dimethacrylate expressed by formula III (m is 1-6), bisphenol-A ethylene oxide-added dimethacrylate expressed by formula IV and tricyclodecanyl dimethylol diacrylabe expressed by formula V.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to an element for controlling the growth direction of nerve fibers and a method for manufacturing the same.

[Conventional technology]

Cultivating neurons in an artificial environment and artificially controlling their growth is useful for elucidating the functions of the brain and nervous system, and for using neurons as information processing elements in computers and other devices. It's important.

Nerve cells do not increase in number except during a certain period, such as during the embryonic period, and because they have long axons and complex dendrites, they are easily damaged when removed from a living body, and there are also problems with survival or the growth of nerve fibers. It has been difficult to culture cells in vitro for several reasons, including the need for small amounts of growth factors.

However, with recent advances in anatomy and tissue culture and the discovery and use of nerve growth factor (NGF), culturing of nerve cells has gradually become easier.

Furthermore, recently, controlling the growth of cultured nerve cells,
For example, it has become possible to control the growth direction of nerve fibers.

The conventional method for controlling the growth direction of nerve fibers is to print proteins such as fibronectin, laminin, and collagen, and polypeptides such as polyornithine and polylysine in a strip shape on a petri dish, and then grow nerve fibers along the strip. [Experimental Cell Research (Exptl, Ce1l Res,) Volume 98.

159-169, 1976), Method for growing nerve fibers in the direction of NGF concentration gradient [Journal of
Cell Biology (J, Ce1lBio1.) 87#
! ! , pp. 546-554.

1980], the method of changing the growth direction to the direction of weak current (Journal of Neuroscience Research)
J, Neurosc i, Res,) vol. 13, 245-
256 pages, 1985].

In addition, a method that combines lithography and ion etching to create an element with a regular groove structure on the surface of quartz glass, and then stretches nerve fibers along the direction of the grooves, has been proposed by Brain Research. R
es,) 446 volumes.

pp. 189-194, 1988, and JP-A-63-11
This is proposed in Publication No. 9754.

[Problem to be solved by the invention]

However, the method of growing nerve fibers along protein or polypeptide prints uses polymers that easily denature, which poses storage problems, and the method using NGF concentration gradients requires extremely sophisticated techniques and is not reproducible. However, the method using a weak current has a problem in that the influence of the t-field on cells has not been fully elucidated. In addition, the method of growing along grooves on the surface of quartz glass involves forming grooves on the surface of quartz glass, and requires advanced techniques such as lithography and ion etching. It is difficult to fabricate devices in this way.

The present invention provides an element for controlling the growth direction of nerve fibers that can be produced stably, easily, and in large quantities, and a method for producing the same.

[Means to solve the problem]

As shown in FIG. 1, the device of the present invention has a surface in which a large number of fine undulations are carved in the portion that comes into contact with living tissue or cells. In the element shown in FIG. 1, the fine undulations are strip grooves, and the strip grooves have a width of 0.1 to 11000 IJ and a depth of 0.1 to 11,000 IJ.
It has a dimension of 1 to 1000 μm, and the grooves are arranged parallel to each other.

The overall shape of the element is not limited to a plate-like shape as shown in Figure 1, but may also be dish-like, spherical, fibrous, cylindrical, or granular.The fine undulations may be not only strips but also paving stones. It's okay,
The strip grooves may be linear, curved, or wavy, and they may be not only parallel but also complex in shape.The cross-sectional shape of the strip grooves may also be arbitrary, such as U-shape, 7-shape, dovetail shape, etc. Something like this is fine.

This device allows nerve fibers to grow oriented along the grooves and the like.

In the present invention, this element is made of an epoxy acrylate represented by the following formula (I) and an epoxy acrylate represented by the following formula (I[l)
(m is an integer from 1 to 6, preferably 4) and the following formula (IV
) OO (n is an integer from 1 to 6, preferably 4) polyethylene glycol diacrylate and bisphenol A ethylene oxide adduct dimethacrylate represented by the following formula (I+) Hs Hs OCHz CHz OCC:CHz (IV) and the following It is molded using one or more photocurable acrylate and/or photocurable methacrylate resins selected from the group consisting of tricyclodecanyl dimethylol diacrylate represented by the formula (V) 0 H Hs M O .

These photocurable acrylate resins and/or photocurable methacrylate resins are used together with a photopolymerization initiator such as l-hydroxycyclohexylphenyl ketone.
- May be used with diluents such as hexane dimethacrylate.

The molding conditions are, for example, room temperature and light intensity of 1,000 to 10.
Light is irradiated for several seconds to several minutes so that the amount of light is 00mJ/cm.

A method of manufacturing the device of the present invention will be explained with reference to FIG.

A metal is deposited on the glass substrate 1 by sputter deposition, vacuum deposition, etc. (FIG. 2(a)), and a photoresist 3 is coated on the metal film 2 using a spin cord (FIG. 2(a)).
b)) Cover the photomask 4 with the desired pattern and expose it to light (Fig. 2(c)). Wash away the exposed areas of the resist with a developer to form the resist pattern 5 (Fig. 2(d)). , remove the metal film 2 with an etching solution (Fig. 2 (e)), wash away the resist pattern 5 remaining on the metal film with a solvent (Fig. 2 (r)), and copy the pattern of the photomask. The stamper 6 you took is completed. Cover the completed stamper 6 with another glass plate 7, pour the synthetic resin 8 between the stamper 6 and the glass 7, and harden the resin with light (Fig. 2 (g)).The glass 7 and the stamper 6
When this is removed, an element 9 (FIG. 2(h)) made of synthetic resin to which the pattern on the surface of the stamper 6 has been transferred is completed.

The metal to be deposited on the glass substrate should be one that has good adhesion to the glass surface (can be easily etched, and has good peelability from the resin after the resin has hardened. Copper, chromium, nickel, etc.) can be used. However, chromium is preferable from the viewpoint of stability.The pattern drawn on the photomask is, for example, 1 to 1000 μm.
Appropriate spacing (as long as it can be observed with a microscope of 1 m),
Any pattern from simple to complex is possible, preferably parallel lines arranged at intervals of 5 to 50 μm, wavy curves, concentric circles or lattice patterns, circuit patterns in which rectangles or circles are connected by parallel straight lines, etc. It is.

〔Example〕

Chromium was sputter-deposited to a film thickness of about 1 μm on a glass substrate measuring 10 cm long, 10 cm wide, and 1 mm thick.

The conditions for sputter deposition at this time are current LA, voltage 35
Argon was used as the gas at 0 V and a pressure of 5 x 10-' Torr, and the amount of metal deposited was 35 cm3/min.

A resist solution prepared by mixing photoresist AZ1350J (trade name manufactured by Hoechst Co., Ltd.) and resist diluent AZ Thinner (trade name manufactured by Hoechst Co., Ltd.) at a weight ratio of LO:3 was then spin-coded to form a resist solution with a thickness of 10 μm. A photomask having equally spaced parallel straight slits was placed over the photomask and exposed to light at a light intensity of 25 to 35 mJ/cm'', followed by cleaning with developer AZ Developer (trade name manufactured by Hoechst) to form a resist pattern. Exposure. Remove the chromium with an etching solution (
After removing it by treating it with an aqueous solution containing 165 g/l of ceric ammonium nitrate and 30 g/l of perchloric acid at a liquid temperature of 40 to 50°C, it was removed using a resist stripper AZ Remover 100 (trade name manufactured by Hoechst). The resist pattern was removed, and a stamper having irregularities at equal intervals of 10 μm was produced.

The thus obtained stamper was covered with another glass plate with a 1 mm thick spacer in between, and bisphenol A ethylene oxide adduct dimethacrylate (molecular weight 16B) was poured into the stamper.
The total amount of light of mm is 3.500~5. The material was cured and molded by irradiation to give an energy density of 10 mJ/cm'', thereby producing an element having a fine groove structure with a groove width of 10 μm2 and an interval of 10 um and a depth of 1 μm.

Using this device, we cultured dorsal root ganglion cells collected from adult mice. The culture solution is Ham F-1
Progesterone (30mM), insulin (5mg/f), and transferrin (I00mM) were added to a 1:1 mixture of 2 medium and Dul-becco MEM medium.
g/j and NGF (7S-NGF 200 ug/j added) at 37°C in a 5% CO□ atmosphere.
After culturing for 48 hours, the growth of nerve fibers was observed under a microscope. As a result, it was observed that nerve fibers were growing along the direction of the microgrooves. If we take the average of five cells that have well-elongated nerve fibers, the ratio of the length of the nerve fibers extending in the direction of the groove to the length that extends in the direction perpendicular to the direction of the groove is 3°8.
This was a larger value than the ratio of 1.0 when cultured on a flat surface without grooves.

〔Effect of the invention〕

The device of the present invention is not made from expensive quartz glass using lithography or ion etching methods that require advanced techniques, as in the past, but instead uses photocurable acrylate, which is commonly used in industrial products. Since it is made by molding/or photocurable methacrylate resin, it can be manufactured at low cost and in large quantities. Also, unlike quartz glass, it is easy to process, so it can be applied to medical devices such as nerve repair materials.

Further, since the method for manufacturing an element of the present invention uses the stamper produced as described above as a mold, any number of elements having the same structure and shape can be stably manufactured, and mass productivity is enhanced.

[Brief explanation of drawings]

FIG. 1 is a perspective view of the device of the present invention, and FIG. 2 is a sectional view showing a method of manufacturing the device of the present invention. Explanation of symbols l...Glass substrate 2...Metal film 3...Photoresist 4...Photomask 5...Resist pattern 6...Stamper 7...Glass plate 8...Synthetic resin (a) (e) (b) (+) (c) Figure (d) (h) Figure

Claims (1)

[Claims] 1. Polyethylene glycol diacrylate represented by the following formula (I) ▲Mathematical formulas, chemical formulas, tables, etc.▼(I) (n is an integer from 1 to 6) and the following formula (II) ▲Mathematical formula , chemical formulas, tables, etc. ▼ (II) Epoxy acrylate represented by and the following formula (III) ▲
There are mathematical formulas, chemical formulas, tables, etc. ▼ (III) (m is an integer from 1 to 6) Polyethylene glycol dimethacrylate and the following formula (IV) ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (IV) One or more types selected from the group consisting of bisphenol A ethylene oxide adduct dimethacrylate and tricyclodecanyl dimethylol diacrylate represented by the following formula (V) ▲Mathematical formula, chemical formula, table, etc.▼(V) An element for controlling the growth direction of nerve fibers, which is molded from photocurable acrylate and/or photocurable methacrylate resin and has a surface in which many fine undulations are carved in the portion that contacts nerve cells. 2. Form a metal vapor deposited film on a glass substrate, form a resist pattern on it, use the resist pattern as a mask to etch the metal vapor deposited film, remove the resist pattern, use the obtained stamper as a mold, and use the synthetic resin as a mold. A method for manufacturing an element for controlling the growth direction of nerve fibers, characterized by molding.