Method of immobilizing cells on solid-phase surface

Abstract

Aspects of the invention can provide a method of immobilizing a chemical compound having the affinity for the cell membrane on the solid-phase surface in a desired pattern. The method of immobilizing a cell in a desired pattern on a solid-phase surface by use of a first chemical compound having an affinity for the cell and can include a step of immobilizing a second chemical compound, which is more easily immobilized on the solid-phase surface than the first chemical compound dose and has a molecular binding site that can bind to the first chemical compound, on the solid-phase surface according to the pattern.

Description

BACKGROUND

Aspects of the invention relates to a method of immobilizing cells on a solid-phase surface according to a desired microscopic pattern. More particularly, the invention relates to a method of immobilizing cells in which a material having affinity for the cells is fixed according to the pattern and then the cells are fixed to the material.

Related art cell immobilization techniques in with which cells can be fixed on a solid-phase surface quickly and precisely has been attracting attention. Living cells that are immobilized can be cultivated on the solid-phase surface. This technique is expected to be applied to such fields as a biosensor and regeneration medicine.

Especially, when the technique is used in the regeneration medicine in order to obtain an alternative to body tissues by multiplying the cells on the solid-phase surface, it is important to fix the cells on a specific position of the solid-phase surface as well as to immobilize the cells without impairing them. An important feature of the tissues and organs is that a necessary cell is deployed in an appropriate position and fulfills its function. Therefore, multiplying cells having different unique finctions and placed in a specific position and building them into a tissue will lead to a development of the body tissue having complex organization structure.

Meanwhile, a cell array, which is cells regularly arrayed and fixed on a substrate, has been under development. The cell array is expected to be used in a manifestation analysis by transforming the cell with a specific gene, assessment of drug toxicity or safety and the like. If it is possible to carry out the manifestation analysis, a drug screening, and the like, on the substrate, there are such advantages as a needful amount of a sample can be reduced, a reaction time can be reduced and a cost of analysis can be cut down.

Methods of immobilizing the cells in a predetermined position of the solid-phase surface has been reported in, for example, Japanese Unexamined Patent Publication No. 2002-355026. A liquid that contains a cell culture control material relating to an adhesive of cells is discharged on a surface of the substrate by a minuscule droplet discharging device. This way, the cell culture control material is patterned in a desired pattern and fixed. Then the cells are cultivated by contacting an area where the culture control material is fixed with a culture solution. In this way, the cells are immobilized on a desired position of the substrate surface.

SUMMARY

When a chemical compound is fixed in an intended pattern on the solid-phase surface by providing a solution including the chemical compound to the solid-phase surface, firstly, the solution has to be provided on the solid-phase surface according to the intended pattern. However, when the chemical compound is made to have an affinity to the cell including the cell culture control material, the chemical compound has to have an affinity for a cell membrane made of lipid. For this reason, the chemical compound often has a hydrocarbon chain which is highly hydrophobic, and at the same time it has a functional group on the opposite side of its end. The functional group plays a role in reacting with the solid-phase surface.

A solvent that can solve such chemical compound and has a high surface tension shows a tendency to contract. Generally, an organic solvent which is highly polarized and has the high surface tension, such as water, uses the tension to minimize a surface free energy. A droplet of the solvent on the solid-phase surface has a relatively low surface free energy and it contracts as maintaining a spherical shape when it evaporates. In this case, for example, when the solution is provided in line pattern by the minuscule droplet discharging device, a shape of the solution will be deformed such that, for example, the solution brakes off into small pieces, by contraction of the solution before the solution is dehydrated. Consequently, the chemical compound, which is a solute, cannot be fixed in clear pattern and the cells immobilized on such chemical compound can not be shaped into the desired pattern.

The present invention is intended to provide a method of immobilizing a chemical compound having the affinity for the cell membrane on the solid-phase surface in a desired pattern.

When cells are immobilized on the solid-phase surface in a desired pattern, before fixing the chemical compound having the affinity for the cell membrane, a compound that is easily fixed on the solid-phase surface in a clear pattern and has a molecular binding site to bind with the chemical compound should be fixed in advance on the solid-phase surface according to the desired pattern. In this way, a chemical compound or a cell having the affinity for the cell can be adequately immobilized according to the desired pattern.

A method of immobilizing a cell of the invention is a method of immobilizing a cell in a desired pattern on a solid-phase surface by use of a first chemical compound having an affinity for the cell and includes a step of immobilizing a second chemical compound, which is more easily immobilized on the solid-phase surface than the first chemical compound dose and has a molecular binding site that can bind to the first chemical compound, on the solid-phase surface according to the pattern. In the method, the second chemical compound may be a compound having the molecular binding site that can bind to the first chemical compound and a substrate binding site that can form a self assembled monolayer film on a solid-phase substrate surface. In the method, the second chemical compound may a compound having the molecular binding site that can bind to the first chemical compound and a polymer molecule part which is physically adsorbed to a solid-phase substrate surface and can form an insoluble thin film. Moreover, in the method, the molecular binding site may be an amino group. In the method, a droplet containing the second chemical compound may be discharged on the solid-phase surface according to the pattern in order to immobilize the second chemical compound.

Furthermore, the method may include a step of immobilizing the second chemical compound on overall the solid-phase surface and a step of inactivating the molecular binding site of the second chemical compound other than the molecular binding site in the pattern. In the method, the molecular binding site of the second chemical compound may be inactivated by irradiating the second chemical compound with a radiant energy ray.

The method may further include a step of forming a cell adhesion preventing film in an area other than the pattern after the second chemical compound is immobilized. Moreover, the method may include a step of forming a cell adhesion preventing film in an area other than the pattern and a step of immobilizing the second chemical compound according to the pattern.

In the method, the cell adhesion preventing film may be formed by discharging a droplet containing a cell adhesion preventing compound in the area other than the pattern. Furthermore, the method may include a step of forming the cell adhesion preventing film on overall the solid-phase surface and a step of removing a cell adhesion preventing compound according to the pattern. In the method, the cell adhesion preventing film may be removed by irradiating the cell adhesion preventing film with a radiant energy ray. Moreover, in the method, the cell adhesion preventing film may include polyethylene glycol, saccharide, polyester and polyisocyanate.

The method may include a step of immobilizing the second chemical compound having the binding site to which a protecting group is bound on overall the solid-phase surface and a step of detaching the protecting group according to the pattern. In the method, the protecting group may be detached by discharging a droplet of the second chemical compound containing a protection removal inducing compound according to the pattern. Furthermore, the method may include a step of removing the second chemical compound by irradiating an area other than the pattern with a radiant energy ray and a step of reacting the second chemical compound left in the pattern with a protection removal inducing compound. In the method, the radiant energy ray may be electromagnetic wave such as ultraviolet rays, electron ray or ion beam. A cell array of the invention can include a cell, which is immobilized by the above-mentioned method.

According to aspects of the invention, a film pattern can be formed accurately according to a pattern in which a cell is immobilized because the second chemical compound which is easily immobilized on the solid-phase surface is firstly immobilized. In this way, the first chemical compound can be accurately immobilized on the second chemical compound. Moreover, the cell can be accurately immobilized according to a desired pattern on the first chemical compound.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be described with reference to the accompanying drawings, wherein like numerals reference like elements, and wherein:

FIGS. 1A through C are process drawings explaining an example of a cell immobilization method according to the invention;

FIGS. 2A through D are process drawings explaining an example of the cell immobilization method according to the invention;

FIGS. 3A through D are process drawings explaining an example of the cell immobilization method according to the invention;

FIGS. 4A through E are process drawings explaining an example of the cell immobilization method according to the invention; and

FIGS. 5A through E are process drawings explaining an example of the cell immobilization method according to the invention.

DETAILED DESCRIPTION OF EMBODIMENTS

An immobilization method according to the invention can include a fixing process in which a second chemical compound is immobilized on the solid-phase surface according to a desired pattern. The second chemical compound has a molecular binding site to bind with a first chemical compound having an affinity for cells and is more easily immobilized on a solid-phase surface than the first chemical compound does.

In the invention, more easily immobilized on the solid-phase surface can mean that things can be immobilized on the solid-phase surface according to the desired pattern without difficulty. Therefore, as the second compound, for example, compounds having a molecular structure which makes the compound quicker to react with the solid-phase surface than the first chemical compound does and being capable of forming a dense thin film can be used.

As an example of such chemical compound, a compound having a substrate binding site which can form a self assembled monolayer (SAM) on a solid substrate surface can be named. When the second chemical compound has the substrate binding site to form the SAM, the second chemical compound will form the dense thin film the solid-phase surface as soon as a solution of the second chemical compound is provided on the solid-phase surface. For this reason, even if a droplet of the solution moves away from its position because of the solution's contraction or a droplet having a line-pattern nearly breaks off, a pattern of the binding site which is connectable to the first chemical compound can be formed before these things happen. The binding site makes the first chemical compound react with the solid substrate surface.

In a case where the solid substrate is metal, thiol group (-SH), disulfide group (-SS-), sulfide group (-S-) can be named as the substrate binding site which can form the SAM. These substrate binding sites, for example, bind with a solid-phase surface which is treated with a gold evaporation and form Au—S binding, and then the SAM is formed.

When the solid-phase surface is made of glass, polymer-resin, semiconductor, metal-oxide and the like, a silane-based coupling agent or a titanate-based coupling agent can be used as the second chemical compound having the substrate binding site to form the SAM. The silane-based coupling agent is generally represented by Y_nSiX_(4-n) (where Y is alkyl group, fluoroalkyl group, vinyl group, amino group, phenyl group or epoxy group and the like, X is alkoxyl group or halogen group and n is a positive integer from 1 to 3). In the case of the silane-based coupling agent, the X part is hydrolyzed and turns into silanol. And then it binds with a hydroxyl group of the solid-phase surface.

Here, as a material for the solid-phase surface in the invention, beside the above-mentioned material, other metals (for example, silver, copper, aluminum, platinum, aluminum oxide, SrTiO₃, LaAlO₃, NdGaO₃ and ZrO₂), silicon (for example, silicon oxide) and polymer resin (for example, polyethylene terephthalate, polycarbonate and the like) can be used. When these materials are used for the solid-phase surface, the SAM can also be adequately formed by using the second chemical compound which has an appropriate finctional group. Furthermore, it is preferred that the solid-phase surface is flat. The solid-phase in the forms of substrate, tape, film and the like are used.

Moreover, in the method according to the invention, as the second chemical compound which is more easily fixed on the solid-phase surface, a compound having a polymer molecule part which is physically adsorbed to the solid-phase surface and can form an insoluble thin film can be used. An adsorption rate of a thin polymer film and a form of the thin polymer film at the time of deposition can be changed by controlling a polymerization degree of the polymer molecule, a concentration of a polymer molecule solution, a temperature of the adsorption and the like. The thin polymer film can be formed with the concentrated solution. Therefore, when the second chemical compound has the polymer molecule part which is physically adsorbed to the solid-phase surface, a deposition film of the second chemical compound can be formed on the solid-phase surface without moving the droplet of the solution away from its position by the solution's contraction as the solution dries out or breaking the line-pattern droplet off.

When the second chemical compound used in the invention has the polymer molecule part, the second chemical compound does not chemically bind to the solid-phase surface but is physically adsorbed to the solid-phase surface and forms the thin film. Since the polymer molecule has a large molecular weight, the second chemical compound can remain on the substrate surface without dissolving in the solution. Therefore, the cells can be immobilized through the first chemical compound if the second chemical compound is provided in the desired pattern in advance.

It should be understood that a structure of the polymer molecule is not especially limited as long as the second chemical compound does not solve out in the solution in an adsorption process of the cells. In a process of making the second chemical compound into the thin film, a water-based buffer solution or the water-based buffer solution containing an organic solvent can be used. As the organic solvent, since polypeptide or protein itself has an especially large amount of amino group on its surface, a special treatment, such as introducing the amino group separately in order to fix the first chemical compound is not necessary. Among many proteins, albumin is most appropriate as the second chemical compound used in the present invention because it is easily coagulated but at the same time it is soluble in neutral and mildly alkaline solution. This means that the albumin can be easily denatured and coagulated with heat, alcohol and the like.

Here, the first chemical compound used in the invention is not especially limited as long as it has the affinity for the cells. Both biosynthetic one and chemically synthetic one can be used. For example, proteins which compose an extracellular matrix (such as collagen, elastin, proteoglycan, fibronectin, laminin and the like) can be used as the first chemical compound. Chemical compounds that can specifically recognize a functional group which is exposed on the cell surface and bind to the functional group can also be used. In addition, antibodies, cytokine, chamical compounds that bind in the way that it is inserted in the cell membrane and the like can be named as the first chemical compound.

Particularly, biocompatible anchor for membrane (BAM) (see, for example, Japanese Unexamined Patent Publication No. 2003-116529) is preferable since it has an aliphatic hydrocarbon group which bines as it is inserted in the cell membrane and it can be immobilized by noncovalent bond without impairing the cells.

The second chemical compound according to the invention can have the above-mentioned binding site which is connectable to the first chemical compound. As such compound, for example, a chemical compound having the following functional group that binds covalently to a functional group included in the first chemical compound as a binding site can be used. The functional group that binds covalently to the first chemical compound includes the amino group, carboxyl group, the disulfide group, the epoxy group, carbodiimide group, maleimide group and the like. Especially when the above-mentioned BAM is used as the first chemical compound, a second chemical compound having the amino group as its binding site is preferred. When the first chemical compound is a biological molecule such as the protein, a material having a biological affinity for the biological molecule such as an antibody, an antigen, and other proteins can be used as the second chemical compound.

In a case where the first chemical compound does not have an appropriate binding site, the functional group such as the amino group, the carboxyl group, the disulfide group, the epoxy group, the carbodiimide group and the maleimide group may be introduced into the first chemical compound. Or a molecule having the biological affinity, such as avidin and biotin may be bound to the first chemical compound. In this case, as the second chemical compound, a compound having a functional group which has affinity for the above-mentioned functional group of the first chemical compound (for example, the amino group, the carboxyl group, the disulfide group, the epoxy group, the carbodiimide group, the maleimide group and the like) may be used. A compound to which molecule having the affinity for the above-mentioned molecule (for example, the avidin, the biotin and the like) is bound may also be used. With such second chemical compound, the first chemical compound can be adequately immobilized. Such feature is included in the invention.

The immobilization process according to the invention is preferably carried out by discharging a droplet containing the second chemical compound on the solid-phase surface. To discharge the droplet, for example, an ink-jet method which can supply a small amount of droplet accurately may be employed. In a way of discharging a solution of the ink-jet method, there are a piezo-jet method using a piezo element, a thermal-jet method using a thermal element and an electrostatic actuator method utilizing an electrostatic force between a diaphragm and an electrode. Any of them can be used in the invention. When a biological material which is sensitive to temperature is contained in the droplet, the piezo-jet method or the electrostatic actuator method is preferably used because the droplet will not be subjected to high temperature.

The droplet including the second chemical compound is discharged according to a pattern in which the cells are immobilized. When an ink-jet device is used, the droplet can be discharged in any pattern you wanted by moving a stage on which the solid phase is placed. The pattern in which the cells are immobilized will be decided in consideration of types and size of the cell and depending on the amount the cell to be cultivated.

Unlike the first chemical compound, it is not necessary for the second chemical compound to have the affinity for the cells. This means that the second chemical compound can be more freely selected compared to the first chemical compound. Therefore, even when the first chemical compound can only dissolve in a solvent unsuitable for discharging as the droplet, a chemical compound which can be solved in a solvent suitable for discharging may be selected as the second chemical compound. This can be discharged on the solid-phase surface. If the second chemical compound is finely patterned, the first chemical compound having the affinity for the cells can be patterned on the solid-phase surface by soaking the solid-phase surface on which the second chemical compound is adsorbed into the solution of the first chemical compound.

The immobilization process according to the invention is preferably performed in the following way. Firstly, the second chemical compound is fixed on the overall solid-phase surface and then the binding site of the second chemical compound can be inactivated in an area other than the pattern in which the cells are immobilized. For example, firstly, the SAM of the second chemical compound is formed on the solid-phase surface. Then, the SAM is irradiated with a radiant energy ray as using a mask having an aperture in the area other than the pattern. In this way, a part of interatomic bonds of the molecule that composes the SAM will be cut and inactivated. As the radiant energy ray, ultraviolet is preferred and a microscopic pattern can be accurately left by irradiating the ultraviolet using the mask. Consequently, the first chemical compound can be accurately immobilized in the desired pattern on the second chemical compound.

In the immobilization method according to the invention, it is preferred that a cell adhesion preventing film is formed in an area of the solid-phase surface other than where the second chemical compound is fixed after the above-described immobilization process. The cell adhesion preventing film includes a cell adhesion preventing compound which does not have the affinity for the cells. The cell adhesion preventing compound is not particularly limited. For example, a chemical compound which has a highly hydrophilic functional group such as a hydroxyl group can be used. As such chemical compound, polyethylene glycol (PEG) and the like can be named.

An SAM having the PEG lined up on its surface can be formed by coupling a functional group that can form the SAM on the solid-phase surface (for example, the thiol group in case where a metal thin film is formed on the solid-phase surface) with an end of the PEG. This can serves as the cell adhesion preventing film. The SAM can be formed by contacting the solid-phase surface with a solution containing the PEG to which the thiol group is bound (hereinafter, calledPEG-thiol) after the second chemical compound is immobilized. The SAM can also be formed by discharging a droplet including the PEG-thiol on an area of the solid-phase surface where the second chemical compound is not immobilized.

By forming the cell adhesion preventing film, it can prevent or reduce that the cell seeps out the pattern and immobilized outside an area where the cell should be immobilized. Therefore, the cell adhesion preventing film can help immobilize the cell more accurately. Furthermore, when the cell is cultivated and multiplied on the solid-phase surface after the cell is immobilized, the cell adhesion preventing film can control an area where the cell should be adhered.

In the immobilization method according to the invention, either immobilization of the second chemical compound or formation of the cell adhesion preventing film may be performed in first. However, it is preferred that the one which can help an accurate patterning is performed first. A case where the formation of the cell adhesion preventing film is performed first will be described below.

The material for the cell adhesion preventing film is described above. Therefore, its explanation will be omitted. A way of forming such cell adhesion preventing film is not especially limited. For example, it can be formed by discharging a droplet containing the cell adhesion preventing compound on the area other than the pattern where the cell is immobilized. The cell adhesion preventing film can also be formed by firstly forming the cell adhesion preventing film on the overall solid-phase surface then removing the film according to the pattern in which the cell is immobilized.

In a case of the latter way, after the cell adhesion preventing film is formed on the overall solid-phase surface in the above-described way, the cell adhesion preventing film may be removed by, for example, irradiating the solid-phase surface with the radiant energy ray, more particularly the ultraviolet, as using a mask having an aperture whose shape is the pattern of the immobilized cell. With the irradiation, an interatomic bond of the cell adhesion preventing compound is broken and the cell adhesion preventing film will be removed.

After the pattern of the cell adhesion preventing film is formed in the above-described way, the second chemical compound is immobilized on the solid-phase surface. A droplet containing the second chemical compound may be discharged on the solid-phase surface according to the pattern in which the cell is immobilized. Or the solid-phase surface on which the cell adhesion preventing film is formed according to the pattern may be contacted with a solution containing the second chemical compound.

Furthermore, the immobilization process according to the invention may include a step of fixing the second chemical compound that has a protecting group bound with the binding site which couples with the first chemical compound, and a step of detaching the protecting group according to the protecting group. The protecting group protects a reactive functional group and it enables the chemical compound to dissolve into various solvents. This helps to pattern more clearly. Moreover, the solid-phase surface can have the high reactive group by using a protection removal inducing agent right before the first chemical compound is reacted. By doing this, reaction with the first chemical compound can progress swiftly and with good reproducibility. Consequently, the first chemical compound can be patterned clearly.

For example, when the binding site coupled with the first chemical compound is the amino group, 9-fluorenylmethoxycarbonyl (Fmoc) is appropriate as the protecting group. When the Fmoc is used to protect, firstly the second chemical compound with the Fmoc is immobilized on the overall solid-phase surface and then the protection is removed according to the pattern in which the cell is immobilized. In the case of the Fmoc, the protection can be removed by contacting with the protection removal inducing agent. To do this, for example, a droplet of the basic solution may be discharged according to the pattern in which the cell is immobilized. As the protection removal inducing agent, a basic solution such as ethanolamine can be used.

It is preferred that the protecting group itself carries out a finction of the cell adhesion preventing film on the solid-phase surface. In this way, an area where the protection is not removed can still serve as the cell adhesion preventing film.

The above-mentioned process of detaching the protecting group may include a step of removing the second chemical compound having the protecting group by irradiating the area other than the pattern in which the cell is immobilized with the radiant energy ray, a step of forming the cell adhesion preventing film on the removed area and a step of reacting the second chemical compound left in the pattern with the protection removal inducing compound.

In the immobilization method according to the invention, after the above-described immobilization process, the first chemical compound can be fixed in the desired pattern by discharging a droplet containing the first-chemical compound according to the second chemical compound pattern. In this case, it is not necessary to discharge the droplet containing the first chemical compound in a beautifully fine line. Therefore, a relatively large amount of the droplet can be discharged and a time to dry the droplet can be extended. In this way, it is possible to secure a longer reaction time between the first chemical compound and the second chemical compound and the first chemical compound will be adequately immobilized.

Moreover, the first chemical compound can also be immobilized by bringing the solid-phase surface in contact with the solution containing the first chemical compound. Since the second chemical compound is already fixed on the solid-phase surface, the first chemical compound is easily immobilized by soaking the solid-phase surface in the solution containing the first chemical compound. In this way, the first chemical compound is easily provided and there is an advantage that a time to react the first chemical compound with the second chemical compound in liquid phase can be secured long enough.

After the first chemical compound is immobilized, the cells can be immobilized by, for example, bringing the solid-phase surface in contact with the solution containing the cells. The solid-phase surface may be soaked in the solution containing the cells or the solution containing the cells may be discharged on the solid-phase surface. Excess cells can be removed by washing since they do not bind to the first chemical compound.

Meanwhile, the present invention also provides a cell array in which the cells are immobilized by the above-described immobilization method. In such cell array, the cells are immobilized alive according to the desired microscopic pattern and they can be cultivated and multiplied. Such cell array is very useful since the cell array can be used, for example, to obtain an alternative to body tissues by multiplying the cells or used as a biosensor for a manifestation analysis.

Although the invention has been fully described by way of the exemplary embodiments described below, it is to be understood that the invention is not limited to the embodiments and various changes and modifications will be apparent to those skilled in the art. Therefore, unless otherwise such changes and modifications depart from the scope of the present invention, they should be construed as being included therein.

The cells are immobilized on the solid-phase surface by the exemplary immobilization method according to the invention. Bovine serum albumin (BSA) is used as the second chemical compound and a compound E290 represented by the following chemical formula (1), which is a kind of the BAM, is used as the first chemical compound. Formula (1)

The BSA has the amino group on its surface and reacts with N-hydroxysuccinimide group of the E290 to fix the E290 on the solid-phase surface.

FIG. 1 is a process drawing explaining the cell immobilization method in a first embodiment. A glass substrate 100 was used as the sold-phase surface. The substrate surface was cleaned by ozone by irradiating the glass substrate surface with UV of 254 nm (10 mW/cm²) for 10 minutes. With the ozone cleaning, a wettability of the surface of the substrate 10 is enhanced and the BSA can be formed in clear pattern. A water contact angle of the glass substrate was less than 10 degrees.

Then, as show in FIG. 1A, a BSA solution 12 (1% phosphate buffer solvent) was discharged on the surface of the substrate 10 in a line & pitch pattern (lines are placed at even intervals) by an ink-jet discharging device. A width of the line was 50 μm and the pitch was 50 μm.

After the BSA solution was dried out, a BAM solution 14 was discharged according to the pattern of the BSA by the ink-jet discharging device as shown in FIG. 1B. In this way the BSA was reacted with the BAM. The BAM solution includes dimethylsulfoxide (DMSO) as solvent, 54 mM of BAM and a little of triethylamine. A sufficient amount of the BAM was discharged so as to cover the whole pattern of the BSA.

After the BAM solution was dried out, the cells are immobilized in line by bringing the BAM pattern in contact with a solution in which a cell 20 is dispersed as show in FIG. 1C.

A reaction between the BAM and the BSA progresses more slowly than the formation of the SAM. The DMSO, which is the solvent of the BMA solution, has a high surface tension and a small contact angle hysteresis. For this reason, a liquid tends to move and the droplet tends to contract before the reaction finishes. This makes it difficult to form a line pattern. In contrast, the BSA solution is a polymer molecule solution and it is possible to control a form of the pattern and a thickness of the film by adjusting a concentration of the solution. An unbreakable and clear pattern can be formed by discharging the BSA solution of 1% high concentration. Therefore, the BAM is also immobilized in the clear pattern. Consequently, the cell is immobilized in the desired pattern.

FIG. 2 is a process drawing explaining the cell immobilization method in a second exemplary embodiment.

In the second exemplary embodiment, firstly, a polyethylene glycol (PEG) thiol [HS—CH₂—(OCH₂CH₂)₇—OH] film 18 was formed as the cell adhesion preventing film on the glass substrate 10 which is treated with a gold evaporation, as show in FIG. 2A. The PEG thiol was an ethanol solution of 1 mM and it was discharged on the area of the substrate 10 other than the pattern in which the cell is immobilized by the ink-jet method. In this way, a thiol group of the PEG thiol reacted with the gold and the SAM was formed. In this embodiment, the ethanol which is highly-volatile is used as the solvent. However, a pattern of the PEG thiol can be formed even in a short time since a thiol compound has a high degree of reactivity to gold.

Then, an amino PEG-thiol [HS—C₂H4—CONH—C₂H₄—(OCH₂CH₂)₇—NH₂] film 12 as the second chemical compound was immobilized on an area where the PEG thiol was not discharged as show in FIG. 2B. The amino PEG-thiol was an ethanol solution of 1 mM and it was discharged on the surface of the substrate 10 by the ink-jet method.

After drying, the BAM solution 14 was discharged according a pattern of the amino PEG-thiol film 12 by the ink-jet discharging device and the amino groups was reacted with the BAM. The BAM solution includes the DMSO as solvent, 54 mM of BAM and a little of triethylamine. A sufficient amount of the BAM was discharged so as to cover the whole pattern of the BSA.

After the BAM solution was dried out, the cells were immobilized in line by bringing the BAM pattern 14 in contact with the solution in which a cell 20 was dispersed as show in FIG. 2D.

In this way, an unbreakable and clear pattern can be formed since the PEG-thiol and the amino PEG-thiol swiftly react with the gold substrate and form the SAM. Therefore, the BAM is immobilized in the clear pattern on the BSA pattern. Consequently, the cell is immobilized in the desired pattern.

Moreover, when the adhesion preventing film which is highly-volatile is formed, the cells will not attach the area where the adhesion preventing film is formed. Therefore, the cells are more precisely immobilized with the adhesion preventing film.

FIG. 3 is a process drawing explaining the cell immobilization method in a third exemplary embodiment.

In the third exemplary embodiment, firstly, the surface of the glass substrate 10 was cleaned by ozone by irradiating the surface with UV of 254 nm (10 mW/cm²) for 10 minutes in the atmosphere. With the ozone cleaning, an organic matter on the glass surface was removed and the clean surface was obtained. After the cleaning, the surface of the substrate was very hydrophilic and a water contact angle of the glass substrate was less than 5 degrees.

Next, the substrate 10 is exposed to an amino-propyl-triethoxy-silane (APTS) vapor for two hours at a temperature of 120° C. and a dense surface membrane 30 of the amino group as shown in FIG. 3A is obtained by a gas phase method. Then, a part of the amino groups on the substrate surface was decomposed by irradiating the surface membrane 30 with an excimer UV of 172 nm through a mask 32 for 15 minutes in a direction indicated by the arrow in the FIG. 3B, and an inactivated area 34 was obtained. The irradiation of the excimer UV was carried out in an atmosphere of N₂ to prevent the amino groups from being oxidized.

As shown in FIG. 3C, the substrate 10 obtained by the above-mentioned processes was soaked in the DMSO solution (54 mM) of the BAM for 5-10 minutes and the BAM 14 was immobilized only an area where the amino group was active.

BAM-solution was dried out, the cells were immobilized in line by bringing the BAM pattern 14 in contact with the solution in which a cell 20 was dispersed as show in FIG. 3D.

According to the above-described method, the film of the amino group can be accurately patterned by irradiating the radiant energy ray as using the mask. Therefore, the BAM is immobilized in the pattern more clearly on the pattern of the amino group. Consequently, the cell is immobilized in the desired pattern.

FIG. 4 is a process drawing explaining the cell immobilization method in a fourth exemplary embodiment.

In the fourth exemplary embodiment, firstly, the surface of the glass substrate 10 treated with the gold evaporation was cleaned by ozone by irradiating the surface with UV of 254 nm (10 mW/cm²) for 10 minutes in the atmosphere. With the ozone cleaning, an organic matter on the glass surface was removed and the clean surface was obtained.

Next, as shown in FIG. 4A, a cell adhesion preventing film 42 of the PEG thiol was formed by soaking the substrate 10 in an ethanol solution of 1 mM PEG thiol for 1-24 hours.

Then, as shown in FIG. 4B, the cell adhesion preventing film 42 was irradiated in the atmosphere of N₂ with the excimer UV of 172 nm through a mask 42 for 15 minutes. An Au—S bond was broken in the irradiated area. The PEG thiol came off from the substrate and the gold surface was exposed again. Then, the substrate 10 was soaked in an ethanol solution of 1 mM amino PEG thiol for 30 minutes as shown in FIG. 4C and a SAM 46 was formed on the exposed gold surface.

After the substrate was dried, the substrate was soaked in the DMSO solution of the BAM and the BAM 14 was immobilized on the SAM 46 which was made of the amino PEG thiol.

After the BAM solution was dried out, the cells were immobilized in line by bringing the BAM pattern in contact with the solution in which a cell 20 was dispersed as show in FIG. 4D.

In this way, the adhesion preventing film and the film of the amino group can be accurately patterned by irradiating the radiant energy ray as using the mask. Therefore, the BAM is immobilized in the pattern more clearly on the pattern of the amino group. Consequently, the cell is immobilized in the desired pattern.

Moreover, the BAM and the amino group can be sufficiently reacted in liquid phase since the substrate is soaked in the BAM solution.

FIG. 5 is a process drawing explaining the cell immobilization method in a fifth exemplary embodiment.

As shown in FIG. 5A, a thin film 52 of N-Fmoc-aminoundecanethiol was formed by soaking the substrate 10 treated with the gold evaporation in an ethanol solution of 1 mM N-Fmoc-aminoundecanethiol.

Then, as shown in FIG. 5B, a protection removal inducing solution 54 (Tris{2-aminoethyl}amine1M/dimethylformamide solution) was provided in line (50 μm) on the substrate by the ink-jet method. And then, an area 56 where the protection of the amino groups was removed and the active amino groups were exposed was obtained as shown in FIG. 5C. The dimethylformamide has a high vapor pressure and its droplet is easily dried off. About 50 μm droplet will dry off in one minute. It takes at least 5 minutes to remove the protection, therefore, the protection removal inducing solution was repeatedly discharged once every minute and the active amino group surface was obtained.

Next, the BAM 14 was immobilized by soaking the substrate 10 in the DMSO solution of the BAM as shown in FIG. 5D.

After the BAM solution was dried out, the cells were immobilized in line by bringing the BAM pattern in contact with the solution in which a cell 20 was dispersed as show in FIG. 5E.

In this way, the BAM can be immobilized in more perfect pattern compared to a case where the BAM solution is directly discharged on the substrate because the basic solution includes a solvent which is more appropriate for the ink-jet method than the BAM solution.

Moreover, a sufficient time to react the BAM with the amino group in liquid phase can be secured since the substrate is soaked in the BAM solution.

Accordingly, while this invention has been described in conjunction with the specific embodiments thereof, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, preferred embodiments of the invention as set forth herein are intended to be illustrative, not limiting. There are changes that may be made without departing from the spirit and scope of the invention.

Claims

1. A method of immobilizing a cell in a desired pattern on a solid-phase surface by use of a first chemical compound having an affinity for the cell, comprising:

immobilizing a second chemical compound on the solid-phase surface according to the pattern, the second chemical compound being more easily immobilized on the solid-phase surface than the first chemical compound and having a molecular binding site that binds to the first chemical compound.

2. The method of immobilizing a cell according to claim 1, the second chemical compound being a compound having the molecular binding site that binds to the first chemical compound and a substrate binding site that forms a self assembled monolayer film on a solid-phase substrate surface.

3. The method of immobilizing a cell according to claim 1, the second chemical compound being a compound having the molecular binding site that binds to the first chemical compound and a polymer molecule part that is physically adsorbed to a solid-phase substrate surface and forms an insoluble thin film.

4. The method of immobilizing a cell according to claim 1, the molecular binding site being an amino group.

5. The method of immobilizing a cell according to claim 1, a droplet containing the second chemical compound being discharged on the solid-phase surface according to the pattern in order to immobilize the second chemical compound.

6. The method of immobilizing a cell according to claim 1, further comprising:

immobilizing the second chemical compound on overall the solid-phase surface; and

inactivating the molecular binding site of the second chemical compound other than the molecular binding site in the pattern.

7. The method of immobilizing a cell according to claim 6, the molecular binding site of the second chemical compound being inactivated by irradiating the second chemical compound with a radiant energy ray.

8. The method of immobilizing a cell according to claim 1, further comprising:

forming a cell adhesion preventing film in an area other than the pattern after the second chemical compound is immobilized.

9. The method of immobilizing a cell according to claim 1, further comprising:

forming a cell adhesion preventing film in an area other than the pattern; and

immobilizing the second chemical compound according to the pattern.

10. The method of immobilizing a cell according to claim 9, the cell adhesion preventing film being formed by discharging a droplet containing a cell adhesion preventing compound in the area other than the pattern.

11. The method of immobilizing a cell according to claim 9, further comprising:

forming the cell adhesion preventing film over all the solid-phase surface; and

removing a cell adhesion preventing compound according to the pattern.

12. The method of immobilizing a cell according to claim 11, the cell adhesion preventing film being removed by irradiating the cell adhesion preventing film with a radiant energy ray.

13. The method of immobilizing a cell according to claim 8, the cell adhesion preventing film including polyethylene glycol, saccharide, polyester and polyisocyanate.

14. The method of immobilizing a cell according to claim 1, further comprising:

immobilizing the second chemical compound having the binding site to which a protecting group is bound on overall the solid-phase surface; and

detaching the protecting group according to the pattern.

15. The method of immobilizing a cell according to claim 14, the protecting group being detached by discharging a droplet of the second chemical compound containing a protection removal inducing compound according to the pattern.

16. The method of immobilizing a cell according to claim 14, further comprising:

removing the second chemical compound by irradiating an area other than the pattern with a radiant energy ray; and

reacting the second chemical compound left in the pattern with a protection removal inducing compound.

17. The method of immobilizing a cell according to claim 7, the radiant energy ray being an electromagnetic wave, such as at least one of ultraviolet rays, electron ray and ion beam.

18. A cell array in which the cell is immobilized by the method of immobilizing a cell according to claim 1.