JP2609073B2 - Cell arrangement control tool and cell arrangement control method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a cell sequence control tool and a cell sequence control method.

[0002]

2. Description of the Related Art In recent years, with rapid advances in cell engineering, LSI technology, medical engineering, and the like, ultra-small biosensors using cells, switching elements, bioreactors, hybrid artificial organs, and even neurocomputers have attracted attention. Gather, these developments are being actively conducted.

[0003] It is difficult to arrange cells as desired and maintain their functions, and this is one barrier to the realization of devices using cells. Cell arrangement control techniques, such as arranging cells as desired to form a circuit network, can be a major key technology for realizing these devices.

[0004]

As an attempt to control the arrangement of cells, there is an example in which fibronectin, which is a cell adhesive protein, is applied using an ink jet printer to form a pattern, and the cells are cultured thereon. However, the resolution is poor and uneven, and is not suitable for fine processing.

[0005] Recently, there has been an attempt to control the direction of nerve cell synapse growth using an artificial concavo-convex surface, but it has not been possible to form a desired arrangement.

[0006]

Means for Solving the Problems In view of such circumstances, the present inventors have conducted intensive studies on a method for easily controlling the arrangement of cells. By culturing cells on the surface of a material having an array pattern, the arrangement of cells can be easily controlled, and a cell array control tool having an array pattern consisting of a cell adhesive surface and a cell non-adhesive surface has a specific The inventors have found that they can be easily manufactured through the steps, and have completed the present invention.

That is, the present invention provides a non-cell-adhesive surface
Cell adhesive hydrophilic polymer with photosensitivity or cell adhesion
Having hydrophilic hydrophilic polymer and two or more azide groups
A cell-adhesive surface made of a photosensitive composition containing a substance , having an array pattern of a cell-adhesive surface and a non-cell-adhesive surface, wherein the cell-adhesive surface is immobilized in a pattern. Claim 1) A non-cell-adhesive hydrophilic polymer having photosensitivity or fine particles on the cell-adhesive surface.
Contains non-adhesive hydrophilic polymer and two or more azide groups
The cell non-adhesive surface made of a photosensitive composition containing a compound to be immobilized in a pattern, having an array pattern comprising a cell adhesive surface and a cell non-adhesive surface, characterized in that The present invention relates to a cell sequence control method (claim 3), which comprises culturing cells using the device (claim 2) and the cell sequence control device according to claim 1 or 2.

[0008]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The cell arrangement controlling device of the present invention has a patterned cell-adhesive surface and a non-cell-adhesive surface formed on the surface of a material serving as the cell arrangement controlling device of the present invention.

[0009] The cell-adhesive surface refers to a charged functional group such as a carboxyl group or an amino group and / or
This refers to a surface into which a cell-adhesive peptide such as RGDS (Arg-Gly-Asp-Ser) is introduced, or a surface to which a polymer having cell adhesive properties is immobilized.

The functional groups such as the carboxyl group and the amino group can be introduced by treating the material surface serving as the alignment control tool of the present invention with radiation such as plasma. In this case, a plastic culture dish, film, tube, or the like can be used as the material.

Specific examples of the polymer having cell adhesion include synthetic polymers having a charge such as polyacrylic acid, polyvinyl sulfate, polystyrene sulfonic acid, and polyallylamine; chondroitin sulfate, dermatan sulfate, dextran sulfate, and keratan. Sulfuric acid, heparan sulfate,
Hyaluronic acid, a polysaccharide having a charge such as chitin, collagen, gelatin, off I Buronekuchin, cell adhesive proteins such as hydro vitronectin, further including synthetic polymers with a fixed cell adhesion proteins and cell adhesion peptides and the like However, the present invention is not limited to these. These may be used alone or in combination of two or more.

The cell non-adhesive surface refers to a hydrophobic surface having a contact angle of 100 degrees or more, or a hydrophilic surface having no charge and a contact angle of 50 degrees or less.

Specific examples of the hydrophobic surface include, but are not limited to, surfaces formed of, for example, polytetrafluoroethylene, silicone, and the like.

Specific examples of the hydrophilic surface having a contact angle of 50 degrees or less include a surface made of a hydrophilic polymer having no charge, for example, polyvinyl alcohol, polyethylene glycol, polyacrylamide, polydimethylacrylamide, polyhydroxyl. Examples include, but are not limited to, ethyl methacrylate, a copolymer of monomers constituting these, cellulose, and the like.

Further, examples of the material that can form the device for controlling the arrangement of cells of the present invention include various plastics, glass, and metals. Materials such as culture dishes and semiconductor substrates that have already been used as devices have been used. Also available.

Next, a method for producing the cell sequence control tool will be described.

First, as a first production method, an array pattern composed of a cell-adhesive surface and a cell-nonadhesive surface is prepared by (1) applying or adsorbing a cell-adhesive hydrophilic polymer having photosensitivity to the cell-adhesive surface. (2) a step of installing a photomask having a desired arrangement pattern on the surface obtained in (1) and pattern-exposing the same; and (3) developing by washing, a cell non-adhesive hydrophilic polymer. A method for forming an image formed on a cell-adhesive surface through a step of forming the image will be described.

[0018] In the first production method, for example, cell non-adhesive hydrophilic polymer, preferably present invention a composition comprising a compound having a hydrophilic high content child and two or more azide groups not having the charge After being present on the cell-adhesive surface serving as a tool for controlling the arrangement of cells, the cells are easily fixed to the cell-adhesive surface by light irradiation. Further, a polymer in which an azide group is directly introduced into the polymer may be used, but the polymer is not required because a special operation for introducing an azide group is unnecessary and an unreacted substance is easily removed during development. It is preferable to use a composition comprising a compound having two or more azide groups.

As the compound having two or more azide groups, for example, a general bis azide compound as shown in Table 1 or an azide polymer having two or more azide groups introduced in one molecule can be used. However, the present invention is not limited to these. Examples of the azide group include carbonyl azide (R-CON ₃ ), sulfonyl azide (R-SO
₂ N ₃ ), aromatic azide

[0020]

Embedded image However, aromatic azide or sulfonyl azide having good stability is preferable. Further, an aromatic azide having an electron-withdrawing substituent such as a nitro group, and an i-line or g-line sensitive bis azide compound are more preferable in that they can be converted to nitrene by light in a longer wavelength range.

[0021]

[Table 1] In the polymer, a nitrene group in which an azide group is converted by light irradiation reacts with a cell-adhesive surface and the polymer by a chemical reaction represented by the following formula, for example, a hydrogen abstraction reaction (1), a double reaction. Addition to bond or insertion into CH bond (2)
, And by performing the coupling reaction (3).

[0022]

Embedded image Since the nitrene group has extremely high reactivity, a bond may be formed by a reaction other than the above-described reaction. In some cases, the above-mentioned reaction occurs between the polymers and cross-linking occurs. In some cases, the polymers are more stably fixed to the cell-adhesive surface. Further, the polymer may not be bound to the cell-adhesive surface as described above, or may be attached and fixed as a film.

As a method for allowing a composition comprising the polymer and a compound having two or more azide groups to be present on a cell-adhesive surface, the composition is dissolved or suspended in a volatile organic solvent such as methanol. This solution is applied or sprayed onto the cell-adhesive surface, and then dried to form a thin layer of the composition on the cell-adhesive surface.The aqueous solution or colloid solution of the composition is brought into contact with the cell-adhesive surface. And a method of adsorbing on a cell-adhesive surface. Among them, the method of casting to form a film using a volatile organic solvent solution is preferable because a homogeneous thin layer can be obtained.

After the compound having two or more azide groups is present on the cell-adhesive surface, the polymer may be present thereon. In this case, also in this specification
Cell-adhesive hydrophilic polymer and two or more azides
When using a photosensitive composition containing a compound having a group
Treated as one mode.

As a light source used for the light irradiation, there are various mercury lamps such as a high-pressure mercury lamp, a low-pressure mercury lamp, an ultra-high-pressure mercury lamp, an excimer laser, and the like. By cutting the short wavelength region, the influence of the short wavelength ultraviolet light on the polymer and the material surface can be reduced. This is particularly preferable when a hydrophilic polymer such as a protein is used.

Since the reaction of the nitrene group is completed in a very short time, the exposure time may be within 5 minutes.

As a method of pattern exposure, there are a method of irradiating light from a photomask having a pattern, and a method of utilizing lithography by excimer laser.

On the other hand, in a second method for producing a cell sequence control tool, in the step (1) of the first method, a photosensitive non-cell-adhesive hydrophilic polymer is applied to a cell-adhesive surface. Instead of adsorbing, the method is the same as the first manufacturing method except that the photosensitive cell-adhesive hydrophilic polymer having a photosensitivity is applied or adsorbed on the non-cell-adhesive surface.

[0029] According to the second method, the high content child and cell non-adhesive comprising a sequence control device of the cells of the present invention the compositions comprising a compound having two or more azide groups having for example the cell adhesiveness After being present on the surface, it is easily fixed to the cell non-adhesive surface by light irradiation. Also before
It is also possible to use a polymer in which an azide group is directly introduced into the polymer
Yes, but no special operation to introduce azide group is required.
And easy removal of unreacted substances during development.
Uses a composition consisting of a compound having two or more azide groups
Is preferred.

By culturing the cells by a conventional method using the cell sequence control tool manufactured as described above, the cell arrangement can be easily controlled, and a high-resolution fine pattern up to the order of μm can be formed. .

The obtained fine pattern is useful for the production of microminiature biosensors, switching elements, bioreactors, hybrid artificial organs, etc., and also for the development of neurocomputers.

Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

Example 1 N, N-dimethylacrylamide monomer (manufactured by Kojin Co., Ltd.) was polymerized in acetone with benzoyl peroxide and N, N-dimethyl-p-toluidine as redox initiators to give poly (N , N-dimethylacrylamide) (hereinafter referred to as PDMAA).

95 parts of the obtained PDMAA (parts by weight, hereinafter the same)
On the other hand, a mixture of 5 parts of sodium 4,4'-diazidostilbene-2,2'-disulfonate, which is a bisazide compound, was dissolved in methanol to prepare a 0.1% (wt%, the same applies hereinafter) solution.

This solution was dropped onto a polystyrene dish for tissue culture (manufactured by CORNING), cast into a film, and air-dried to form a film having a thickness of several tens of nm. A photomask having a pair of slits each having an opening and a non-opening and having a width of 250 μm as shown in Fig. 7 was set, and pattern exposure was performed using a high-pressure mercury lamp for 30 seconds. FIG. 3 is a sketch drawing of a photo of a photomask.

Next, the plate was sufficiently washed with methanol and water and developed to obtain a Petri dish in which a fine pattern composed of PDMAA and the Petri dish surface was formed.

The petri dish thus obtained was seeded with bovine vascular endothelial cells, and DMEM (Dulbe) containing 15% calf serum (FCS) was added.
cco's Modified Eagle's Medium)
When cultured in a CO ₂ incubator at ℃, the endothelial cells selectively expanded and proliferated only in the PDMAA non-fixed portion (non-exposed portion), and the cell sequence patterns shown in FIGS. 1 and 2 were obtained. FIG. 1 is a sketch drawing of a photograph of the arrangement pattern of the stained cells (the magnification is the same as the original photograph of FIG. 3),
FIG. 2 is a sketch diagram of a photograph further enlarged than the photograph on which FIG. 1 is based.

Example 2 A 0.1% methanol solution of PDMAA containing a bisazide compound prepared in the same manner as in Example 1 was dropped on a polystyrene dish, cast, formed into a film, and air-dried. A Petri dish with PDMAA fixed by light (hereinafter referred to as PDMAA Petri dish) was obtained.

A fibronectin and a copolymer comprising 80 parts of N, N-dimethylacrylamide and 20 parts of acryloxysuccinimide (manufactured by Kokusan Kagaku) were mixed with a phosphate buffer (pH 8.
5), and N, N- immobilized fibronectin
A dimethylacrylamide copolymer (hereinafter, referred to as FN-PDMAA) was obtained.

A mixture of 95 parts of FN-PDMAA and 5 parts of a bisazide compound was dissolved in methanol to prepare a 0.1% solution.

The obtained solution was cast on a PDMAA petri dish and air-dried to form a film having a thickness of several tens of nm. Then, a photomask was set, and pattern exposure was performed for 30 seconds using a high-pressure mercury lamp.

Next, the plate was sufficiently washed with methanol and water and developed to obtain a Petri dish in which a fine pattern composed of FN-PDMAA and PDMAA Petri dishes was formed.

When bovine vascular endothelial cells were cultured in the same manner as in Example 1 using the petri dish thus obtained, the endothelial cells were selectively applied only to the FN-PDMAA-fixed portion (exposed portion). It extended and proliferated, and an array pattern by cells was obtained.

[0044]

The cell sequence control device of the present invention has good selectivity for the presence or absence of cell attachment.
The cell arrangement can be easily and precisely controlled by culturing in the same manner as the conventional cell culture, and an extremely fine and high-resolution cell arrangement pattern can be easily formed.

The present invention greatly contributes to the development of ultra-small biosensors, switching elements, hybrid artificial organs, bioreactors, neurocomputers and the like that utilize various cell functions. The present invention can also be applied to the study of cell functions such as information transmission between cells.

[Brief description of the drawings]

FIG. 1 is a sketch drawing of a photograph of an array pattern of stained cells.

FIG. 2 is a sketch drawing of a photograph further enlarged than the photograph on which FIG. 1 is based;

FIG. 3 is a sketch drawing of a photo of a photomask (the magnification is the same as the photo on which FIG. 1 is based).

Claims (3)

(57) [Claims] Claims: 1. A cell-adhesive hydrophilic polymer or a cell-adhesive hydrophilic polymer having photosensitivity on a non-cell-adhesive surface .
And photosensitive group containing compounds having two or more azide groups
Cell adhesive surface consisting Narubutsu is immobilized in a pattern, sequence control device of cells, characterized by having the arrangement pattern composed of a cell adhesive surface and a cell non-adhesive surface. 2. A non-cell-adhesive hydrophilic polymer or a non-cell-adhesive hydrophilic polymer having photosensitivity on a cell-adhesive surface.
Containing a compound having two or more azide groups
A cell sequence control device having an array pattern comprising a cell adhesive surface and a cell non-adhesive surface, wherein the cell non-adhesive surface made of the composition is immobilized in a pattern. 3. A cell sequence control method, comprising culturing cells using the cell sequence control tool according to claim 1.