JP4761731B2 - Method for producing substrate on which cells are immobilized and substrate - Google Patents

Description

  The present invention relates to a method for producing a substrate on which cells are immobilized, and a substrate that can be used in the method.

  In recent years, cells have been cultured in a so-called cell array in which cells are adhered to a defined area on a culture substrate. In particular, in a wide range of fields such as tissue engineering, cell-based sensor development, and basic research on cell biology, using cell arrays patterned with two or more chemical species that are adherent and non-adhesive to cells Therefore, establishment of a technique (cell patterning) for defining an adhesion region and a non-adhesion region of cells is required. Among them, a technology that converts (switches) cell adhesion in a specific region on a culture substrate in response to external stimuli such as heat, electricity, and light is attracting attention as a next-generation culture technology. This technology can be applied as a cell array production technique for drug / toxic substance screening as a research tool for cell-to-cell information transmission between allogeneic and heterogeneous cells.

  In particular, in technology that converts cell adhesion in response to external stimuli, which is being developed in recent years, functional materials that convert surface chemical species such as phase transitions, oxidation / reduction, and chemical reactions in response to external stimuli are available. It is used. Non-patent documents 1 to 8 disclose cell adhesion switching techniques using such functional materials, for example. Non-Patent Documents 1 and 2 propose a technique for switching between cell adhesion / non-adhesion using a temperature-responsive polymer film. Non-Patent Documents 3 to 6 propose techniques for attaching and detaching cells using a monomolecular film having electrochemical activity. Non-Patent Document 7 proposes a technique in which a microelectrode is used to locally generate an oxidant to denature cell adhesion-inhibiting bovine serum albumin and convert it to cell adhesion. Non-Patent Document 8 proposes a technique for converting cell adhesion using a photoresponsive polymer having spiropyran as a photochromic compound in the side chain.

  However, in the techniques described in Non-Patent Documents 1 to 6, in order to apply heat and electric potential to the entire substrate, it is necessary to define a region where cell adhesiveness can be converted in advance, and the manufacturing process of the culture substrate is complicated. there were. Moreover, since the area | region which can convert adhesiveness is prescribed | regulated previously, after observing the mode of adhesion of a cell under a microscope after cell seeding, the conversion area could not be changed.

  On the other hand, according to the techniques described in Non-Patent Documents 7 and 8, cell adhesion patterning can be newly formed after cell seeding, or the size thereof can be changed. However, the technique described in Non-Patent Document 7 requires a microelectrode to generate an oxidant locally, and it is difficult to maintain a sterile environment of cells in order to bring the microelectrode closer to the substrate. Conceivable. Moreover, there is a concern about the influence of the generated oxidizing agent on the cells. Furthermore, since the diffusion range of the oxidizing agent determines the resolution, it is expected that it is difficult to form a fine pattern.

On the other hand, in the technique described in Non-Patent Document 8, since the photoisomerization efficiency of spiropyran is not good, the cell adhesiveness change efficiency is poor and has not reached the practical level. Moreover, since a high output light source is required for the photoisomerization reaction, it is not practical in terms of cost and operation technique. In addition, since the compound used in this technique is reversibly isomerized by light, there is a concern that it will return to its original structure by absorption of visible light or the like over time.
Makromol. Chem. Rapid.Comm 11: 571-576 (1990) Biomaterials 23: 1121-1130 (2002) Proc. Natl. Acad. Sci. USA 98: 5992-5996 (2001) ChemBioChem 7: 590-3 (2001), J. Amer. Chem. Soc. 125: 14994-14995 (2003) J. Amer. Chem. Soc. 125: 2366-2367 (2003) Langmuir 20: 16-19 (2004) Polymer Preprints, Japan 53: 1933 (2004)

  Therefore, an object of the present invention is to provide a method for producing a substrate on which cells are immobilized, in which a new cell adhesion patterning can be formed and resized after cell seeding, particularly under cell culture.

Means for achieving the object is as follows.
[Claim 1] A method for producing a substrate on which cells are immobilized, comprising:
Immobilizing a compound whose terminal functional group is protected by a photodegradable protecting group on a substrate,
Removing at least part of the photodegradable protecting group by light irradiation to expose at least part of the terminal functional group;
The method, wherein cells are adsorbed to at least a part of the exposed terminal functional group.
[Claim 2] After the cell adsorption, at least a part of the photodegradable protective group that is not detached by the light irradiation is detached by the light irradiation to expose at least a part of the terminal functional group,
The method according to claim 1, wherein a cell that is the same or different from the cell is adsorbed to at least a part of the exposed terminal functional group.
[Claim 3] A method for producing a substrate on which cells are immobilized, comprising:
Immobilizing a compound whose terminal functional group is protected by a photodegradable protecting group on a substrate,
Adsorbing a cell adhesion inhibitor on at least a part of the photodegradable protecting group;
Removing at least part of the photodegradable protecting group adsorbed by the cell adhesion inhibitor by light irradiation to expose at least part of the terminal functional group;
The method, wherein cells are adsorbed to at least a part of the exposed terminal functional group.
[Claim 4] After the cell adsorption, at least a part of the photodegradable protective group adsorbed by the cell adhesion-inhibiting substance that is not detached by the light irradiation is removed by light irradiation, and at least a part of the terminal functional group is removed. To expose
The method according to claim 3, wherein a cell that is the same or different from the cell is adsorbed to at least a part of the exposed terminal functional group.
[5] A method for producing a substrate on which cells are immobilized,
Immobilizing a compound whose terminal functional group is protected by a photodegradable protecting group on a substrate,
Adsorbing a cell adhesion inhibitor on at least a part of the photodegradable protecting group;
Removing at least part of the photodegradable protecting group adsorbed by the cell adhesion inhibitor by light irradiation to expose at least part of the terminal functional group;
Adsorbing a cell adhesion promoting substance on at least a part of the exposed terminal functional group;
The method described above, wherein cells are adsorbed to at least a part of the cell adhesion promoting substance.
[Claim 6] After the cell adsorption, at least a part of the photodegradable protective group adsorbed by the cell adhesion-inhibiting substance that is not detached by the light irradiation is removed by light irradiation, and at least a part of the terminal functional group is removed. To expose
Adsorbing a cell adhesion promoting substance that is the same or different from the cell adhesion promoting substance on at least a part of the exposed terminal functional group;
The method according to claim 5, wherein cells that are the same or different from the cells are adsorbed to at least a part of the cell adhesion promoting substance.
[7] The method according to any one of [1] to [6], wherein the light irradiation is performed through a photomask.
[8] The method according to any one of [1] to [7], wherein the terminal functional group is a carboxyl group.
[9] The method according to any one of [1] to [8], wherein the substrate on which the cells are immobilized is a cell array.
[10] A substrate on which a compound having a terminal functional group protected by a photodegradable protecting group is immobilized on a substrate, wherein a cell adhesion inhibitor is adsorbed on at least a part of the photodegradable protecting group. The substrate.

  According to the method for producing a substrate on which cells of the present invention are immobilized, new cell adhesion patterning and size change can be easily performed after cell seeding, particularly under cell culture.

Hereinafter, the present invention will be described in more detail.

A first aspect of the present invention is a method for producing a substrate on which cells are immobilized, wherein a compound having a terminal functional group protected by a photodegradable protecting group is immobilized on the substrate, and the photodegradable by light irradiation. In this method, at least a part of the protecting group is removed to expose at least a part of the terminal functional group, and cells are adsorbed to at least a part of the exposed terminal functional group.
According to a second aspect of the present invention, there is provided a method for producing a substrate on which a cell is immobilized, wherein a compound having a terminal functional group protected by a photodegradable protecting group is immobilized on the substrate, At least a part of the cell adhesion inhibitor is adsorbed by at least a part, and at least a part of the photodegradable protective group adsorbed by the cell adhesion inhibitor by light irradiation is removed to expose at least a part of the terminal functional group, the exposure The method is characterized in that cells are adsorbed to at least a part of the terminal functional group.
A third aspect of the present invention is a method for producing a substrate having cells immobilized thereon, wherein a compound having a terminal functional group protected by a photodegradable protecting group is immobilized on the substrate, and the photodegradable protecting group is At least a part of the cell adhesion inhibitor is adsorbed by at least a part, and at least a part of the photodegradable protective group adsorbed by the cell adhesion inhibitor by light irradiation is removed to expose at least a part of the terminal functional group, In this method, the cell adhesion promoting substance is adsorbed on at least a part of the terminal functional group, and the cells are adsorbed on at least a part of the cell adhesion promoting substance.
Hereinafter, the first, second, and third aspects of the present invention are also referred to as the method of the present invention.

In the method of the present invention, first, a compound having a terminal functional group protected by a photodegradable protective group (hereinafter, also referred to as “photodegradable protective group-containing compound”) is immobilized on a substrate.
The photodegradable protective group-containing compound is a compound in which a terminal functional group is protected by a photodegradable protective group, and the photodegradable protective group is released by irradiation with light to expose the terminal functional group.
The photodegradable protecting group refers to any group that can be eliminated by light irradiation, such as a nitrobenzyl group, a nitrophenylethyl ester group (NPE), a dimethoxynitrobenzyl ester group (DMNB), or a bromohydroxycoumarin (Bhc) group. , Dimethoxybenzoin group, 2-nitropiperonyloxycarbonyl (NPOC) group, 2-nitroveratryloxycarbonyl (NVOC) group, α-methyl-2-nitropiperonyloxycarbonyl (MeNPOC) group, α-methyl 2-nitroveratryloxycarbonyl (MeNVOC) group, 2,6-dinitrobenzyloxycarbonyl (DNBOC) group, α-methyl-2,6-dinitrobenzyloxycarbonyl (MeDNBOC) group, 1- (2-nitrophenyl) ) Ethyloxycarbonyl (NPEOC) group, 1-methyl- 1- (2-nitrophenyl) ethyloxycarbonyl (MeNPEOC) group, 9-anthracenylmethyloxycarbonyl (ANMOC) group, 1-pyrenylmethyloxycarbonyl (PYMOC) group, 3'-methoxybenzoinyloxycarbonyl (MBOC) group, 3 ', 5'-dimethoxybenzoyloxycarbonyl (DMBOC) group, 7-nitroindolinyloxycarbonyl (NIOC) group, 5,7-dinitroindolinyloxycarbonyl (DNIOC) group, 2-anthracoxy Nonylmethyloxycarbonyl (AQMOC) group, α, α-dimethyl-3,5-dimethoxybenzyloxycarbonyl group, 5-bromo-7-nitroindolinyloxycarbonyl (BNIOC) group and the like can be mentioned. -Nitrobenzyl derivative skeleton The group having is preferable from the viewpoints of raw material cost and ease of synthesis. In addition, since a photodegradable protective group-containing compound having a group having a 2-nitrobenzyl derivative skeleton as a photodegradable protective group does not undergo photodegradation in the degree of indoor lighting such as ordinary fluorescent lamps and incandescent lamps, There is also an advantage that the substrate on which the compound is immobilized is easy to handle.

In the second and third aspects of the present invention, as will be described later, the photodegradable protective group is used to adsorb the cell adhesion inhibitory substance to at least a part of the photodegradable protective group. Is preferably selected from the viewpoint of good adsorption.
On the other hand, in the first aspect of the present invention, as will be described later, at least a part of the photodegradable protective group is removed by light irradiation to remove a terminal functional group, and at least one of the exposed terminal functional groups is removed. Cell patterning is formed by adsorbing cells to the part. Therefore, in the first embodiment, in order to selectively adsorb cells to the exposed terminal functional group, it is preferable that the photodegradable protective group is not adsorbable to the cells to be used.

In the photodegradable protective group-containing compound, the terminal functional group protected by the photodegradable protective group may be a carboxyl group, an amino group, a sulfo group, a thiol group, a hydroxyl group, or a phosphate group, It is preferable that
In the first and second embodiments of the present invention, cells are adsorbed to the terminal functional group that is deprotected and exposed by light irradiation. Therefore, in the first and second embodiments, the terminal functional group is preferably one that adsorbs the cells to be used well.
On the other hand, in the third aspect of the present invention, since the cell adhesion promoting substance is adsorbed on the terminal functional group, the terminal functional group preferably adsorbs the cell adhesion promoting substance used well.

  The photodegradable protective group-containing compound is preferably a compound that forms a self-assembled monolayer. If the photodegradable protective group-containing compound forms a self-assembled monolayer after immobilization on the substrate, the photodegradable protective group is aligned on the outermost surface of the substrate, and then the protective group is released by light irradiation. Thus, the terminal functional group can be exposed on the outermost surface of the substrate. On such a substrate, if the cells are adsorbed after adsorbing the cells in the first and second embodiments and adsorbing the cell adhesion promoting substance in the third embodiment, the substrate having the cells adsorbed on the outermost surface is obtained. Obtainable. Examples of the compound that forms a self-assembled monolayer include alkylsiloxane compounds, alkanethiol compounds, and acrylic compounds.

  The photodegradable protecting group-containing compound used in the present invention can be an alkylsiloxane compound in which a terminal functional group is protected by a photodegradable protecting group. If the photodegradable protective group-containing compound is an alkylsiloxane compound, it can be easily immobilized on a glass substrate. The silane part of such an alkylsiloxane-based compound can be trihalolosilane or trialkoxysilane. The alkyl part of the alkylsiloxane compound can be a linear or branched saturated alkyl chain or an unsaturated alkyl chain. In view of excellent packing of the self-assembled film immobilized on the substrate surface, the alkyl part is preferably a linear alkyl chain. The number of carbon atoms can be an integer in the range of, for example, 1 to 20, and is preferably an integer in the range of 4 to 20 in consideration of the packing of the self-assembled film and the availability of raw materials.

  Specifically, examples of the alkylsiloxane compound in which the terminal functional group is protected by a photodegradable protecting group include the following silane compounds in which a carboxyl group is protected by o-nitrobenzyl ester. For the synthesis method, Chem. Lett. 2000, 228-229 can be referred to.

  In the silane compound, a carboxyl group is derived as a terminal functional group by eliminating a nitrobenzyl group which is a photodegradable protective group by light irradiation. Specific examples of such compounds include 5-trichlorosilylpentanoic acid (2-nitrophenyl) ethyl ester shown below.

  On the other hand, examples of the silane compound in which an amino group is derived as a terminal functional group by light irradiation include compounds represented by the following general formula [1].

In general formula [1], X represents an alkoxy group or a halogen atom, and is preferably an alkoxy group.
The alkoxy group represented by X is not particularly limited, but is preferably an alkoxy group having 1 to 6 carbon atoms, specifically, a methoxy group, an ethoxy group, a propoxy group, An isopropoxy group, a butoxy group, an isobutoxy group, an s-butoxy group, a t-butoxy group, an n-pentyloxy group, an n-hexyloxy group, and the like can be mentioned. Among these, a methoxy group and an ethoxy group are particularly preferable. Moreover, as a halogen atom represented by X, a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, etc. can be mentioned.

In general formula [1], R ¹ represents an alkyl group. The alkyl group represented by R ¹ may be linear or branched, may have an unsaturated bond, may be an alkoxy group such as a methoxy group or an ethoxy group, May have a substituent such as an acyl group such as a formyl group and an acetyl group. The alkyl group is not particularly limited, but preferably has 1 to 6 carbon atoms. Specifically, a methyl group, an ethyl group, an n-propyl group, an isopropyl group, a butyl group, alkyl groups such as s-butyl group, t-butyl group, n-pentyl group, isopentyl group, neopentyl group, n-hexyl group, vinyl group, allyl group, 2-propenyl group, 2-butenyl group, 2-pentenyl group Specific examples include alkenyl groups such as 2-heptynyl groups and alkynyl groups such as propargyl groups, and among these, methyl groups are particularly preferable.

  In the general formula [1], m represents an integer of 1 to 3, and is preferably as large as possible because it can be easily fixed to a substrate. Moreover, in general formula [1], n represents an integer, and is preferably an integer of 1 to 20 and more preferably an integer of 2 to 15 from the viewpoint of easy availability of starting materials.

  In general formula [1], Y represents a photodegradable protecting group. As described above, the photodegradable protecting group refers to any group that can be removed by light irradiation, such as a group having a 2-nitrobenzyl derivative skeleton, a dimethoxybenzoin group, 2-nitropiperonyloxycarbonyl (NPOC). Group, 2-nitroveratryloxycarbonyl (NVOC) group, α-methyl-2-nitropiperonyloxycarbonyl (MeNPOC) group, α-methyl-2-nitroveratryloxycarbonyl (MeNVOC) group, 2,6 -Dinitrobenzyloxycarbonyl (DNBOC) group, α-methyl-2,6-dinitrobenzyloxycarbonyl (MeDNBOC) group, 1- (2-nitrophenyl) ethyloxycarbonyl (NPEOC) group, 1-methyl-1- ( 2-Nitrophenyl) ethyloxycarbonyl (MeNPEOC) group, 9-an Rasenylmethyloxycarbonyl (ANMOC) group, 1-pyrenylmethyloxycarbonyl (PYMOC) group, 3'-methoxybenzoinyloxycarbonyl (MBOC) group, 3 ', 5'-dimethoxybenzoyloxycarbonyl (DMBOC) group 7-nitroindolinyloxycarbonyl (NIOC) group, 5,7-dinitroindolinyloxycarbonyl (DNIOC) group, 2-anthraquinonylmethyloxycarbonyl (AQMOC) group, α, α-dimethyl-3,5- A dimethoxybenzyloxycarbonyl group, a 5-bromo-7-nitroindolinyloxycarbonyl (BNIOC) group, a bromohydroxycoumarin (Bhc) group, and the like can be mentioned. In the silane compound represented by the following general formula [2] 2-nitrobenzyl induction Particularly preferred groups having a skeleton.

Among the silane compounds represented by the general formula [1], the silane compound represented by the general formula [2] is preferable. In the general formula [2], R ² represents a hydrogen atom or an alkyl group, and R ^The alkyl group represented by ² may be linear or branched, may have an unsaturated bond, an alkoxy group such as a methoxy group or an ethoxy group, You may have substituents, such as acyl groups, such as a formyl group and an acetyl group. The alkyl group is not limited in carbon number, but preferably has 1 to 6 carbon atoms. Specifically, methyl group, ethyl group, n-propyl group, isopropyl group, butyl group, s Alkyl groups such as -butyl group, t-butyl group, n-pentyl group, isopentyl group, neopentyl group, n-hexyl group, vinyl group, allyl group, 2-propenyl group, 2-butenyl group, 2-pentenyl group An alkenyl group such as 2-heptynyl group, an alkynyl group such as propargyl group, and the like can be specifically exemplified. Among these, a methyl group can be particularly preferred.

In general formula [2], R ³ and R ⁴ each independently represents a hydrogen atom or an alkoxy group. That is, R ³ and R ⁴ may be the same substituent or different substituents, but are preferably the same group, and are preferably alkoxy groups. The alkoxy group represented by R ³ and R ⁴ is not particularly limited, but is preferably an alkoxy group having 1 to 6 carbon atoms, specifically, a methoxy group or an ethoxy group. , Propoxy group, isopropoxy group, butoxy group, isobutoxy group, s-butoxy group, t-butoxy group, n-pentyloxy group, n-hexyloxy group, etc., among which methoxy group and ethoxy group are particularly preferable preferable.

  On the other hand, examples of the silane compound in which a sulfo group is derived as a terminal functional group by light irradiation include compounds represented by the following general formula [3].

In general formula [3], X represents an alkoxy group or a halogen atom, Y represents a photodegradable protecting group, and R ¹ represents an alkyl group. m represents an integer of 1 to 3, and n represents an integer. X, Y, for R ¹ in the general formula [3], the same X, Y, and R ¹ in formula (1). M and n in the general formula [3] are the same as m and n in the general formula [1].

Among the silane compounds represented by the general formula [3], the silane compound represented by the general formula [4] is preferable. In the general formula [4], R ² represents a hydrogen atom or an alkyl group, and R ³ and R ⁴ each independently represents a hydrogen atom or an alkoxy group. For R ^2, R ^3, R ⁴ in the general formula [4] is the same as R ^2, R ^3, R ⁴ in the general formula (2).

  On the other hand, examples of the compound in which a thiol group is derived as a terminal functional group by light irradiation include compounds represented by the following general formula [5].

In general formula [5], X represents an alkoxy group or a halogen atom, Y represents a photodegradable protecting group, and R ¹ represents an alkyl group. m represents an integer of 1 to 3, and n represents an integer. X, Y, for R ¹ in the general formula [5], the same X, Y, and R ¹ in formula (1). M and n in the general formula [5] are the same as m and n in the general formula [1].

Among the silane compounds represented by the general formula [5], a silane compound represented by the general formula [6] is preferable. In the general formula [6], R ² represents a hydrogen atom or an alkyl group, and R ³ and R ⁴ each independently represents a hydrogen atom or an alkoxy group. For R ^2, R ^3, R ⁴ in the general formula [6] is the same as R ^2, R ^3, R ⁴ in the general formula (2).

  Examples of the silane compound in which a phosphate group is derived as a terminal functional group by light irradiation include compounds represented by the following general formula [9].

In the general formula [9], X represents an alkoxy group or a halogen atom, Y represents a photodegradable protecting group, W represents Y or a hydrogen atom, and R ¹ represents an alkyl group. m represents an integer of 1 to 3, n represents an integer, and l represents 0 or 1. X, Y, for R ¹ in the general formula [9], the same X, Y, and R ¹ in formula (1). M and n in the general formula [9] are the same as m and n in the general formula [1].

Among the silane compounds represented by the general formula [9], the silane compounds represented by the general formula [10] or the general formula [11] are preferable. In the general formulas [10] and [11], R ² Represents a hydrogen atom or an alkyl group, and R ³ and R ⁴ each independently represent a hydrogen atom or an alkoxy group. For R ^2, R ^3, R ⁴ in the general formula [10] and [11] is the same as R ^2, R ^3, R ⁴ in the general formula (2).

  Among the silane compounds represented by the general formulas [1] to [6] and the general formulas [9] to [11] (including the case where m is 0), the general formula [2] and the general formula [4] The silane compounds represented by the general formula [6], the general formula [10], and the general formula [11] are preferable, and among these, a compound in which m is an integer of 1 to 3 is preferable. 2-nitrophenyl) ethyl-N- (3- (triethoxysilyl) propyl) carbamate (specific example 1), 1- (2-nitrophenyl) ethyl-N- (3- (trimethoxysilyl) propyl) carbamate ( Specific example 2), 1- (2-nitrophenyl) ethyl-N- (3- (methyldiethoxysilyl) propyl) carbamate (specific example 3), 1- (4,5-dimethoxy-2-nitrophenyl) ethyl -N- (3- (Triethoxy Silyl) propyl) carbamate (specific example 4), 1- (2-nitrophenyl) ethyl-N- (11-triethoxysilyl) propyl) carbamate (specific example 5), 2-nitrobenzyl-4- (trimethoxysilyl) ) Butanesulfonate (specific example 6), 2-nitrobenzyl-4- (triethoxysilyl) butanesulfonate (specific example 7), 2-nitrobenzyl-3- (trimethoxysilyl) propyl sulfide (specific example 8), etc. Can be mentioned.

  JP, 2003-321479, A can be referred to for details, such as a synthesis method of the above-mentioned silane compound.

  When using an alkylsiloxane compound having a terminal functional group protected by a photodegradable protective group as the photodegradable protective group-containing compound, the substrate on which the compound is immobilized is preferably a glass substrate. Immobilization of the alkylsiloxane compound on the glass substrate can be performed by a known method. For example, the alkylsiloxane compound can be immobilized on the glass substrate by dissolving the alkylsiloxane compound in a suitable solvent, coating the glass substrate, and coupling the alkylsiloxane compound to the substrate. Examples of the coating means include application, spraying, dipping, etc. Among them, dipping is preferable. Immobilization can be performed by room temperature treatment, and it is preferable to perform reflux treatment. The glass substrate can be pretreated before immobilization. The pretreatment can be performed by coating an acidic solution on the substrate. Examples of the acidic solution include sulfuric acid, hydrochloric acid, nitric acid, hydrogen peroxide, and the like. These may be used alone or in combination of two or more, but the combined use of sulfuric acid and hydrogen peroxide is preferable. Examples of the coating means include coating, spraying, and dipping. By this pretreatment, a hydrophilic group (silanol group) can be formed on the substrate.

  Furthermore, in the present invention, when a substrate having a metal surface is used, as a photodegradable protective group-containing compound, an alkanethiol whose terminal functional group is protected by a photodegradable protective group, or a disulfide bond (—S— Sulfur-containing compounds such as compounds having S-) can be used. These sulfur-containing compounds can be immobilized on a metal substrate by a metal-sulfur bond.

  Examples of such a sulfur-containing compound include the compounds shown below.

In the formula, X ′ is a group that induces a target functional group, and preferably a carbamate group, a carboxylic acid ester group, a carbonate group, an ether group, a thiocarbonate group, a thioether group, or a sulfonic acid ester group. These groups are present in the thiol compound or disulfide compound in the following arrangement, and the functional group described in the right column is derived by elimination of the photoreleasable protecting group (Y ′). In the table below, z represents a bond bonded to Z, and y represents a bond bonded to Y ′.

Y ′ is a photoreleasable protecting group, and has a property of protecting X ′ in a predetermined reaction step and leaving by light irradiation. As such a photolabile protecting group, a photolabile protecting group represented by the following formula [15] is preferably exemplified.

R ¹¹ is hydrogen or a methyl group,
b is an integer of 1-4, preferably an integer of 1-2,
R ¹² is hydrogen or a methoxy group, and when b is 2 or more, the two R ¹² may jointly form a ring that may contain oxygen (for example, a methylenedioxy group).
Accordingly, preferable examples of the photodetachable protecting group include those having the following structures.

The photoreleasable protecting group Y ′ as described above is eliminated from X ′ by light irradiation to induce the groups shown in the above table. When Y ′ is desorbed, part of X ′ may be desorbed together. For example, in the above table, when an amine group is derived from a carbamate group, the COO group in the carbamate group is also removed together with the photoremovable protective group Y ′.
Z is a divalent hydrocarbon group which may contain a hetero atom, and is not particularly limited as long as the formation of the monomolecular film is not impaired.
Preferably _{- (CH 2) c -,} - (CH 2 -CH 2 -O) d -,

Chosen from. Here, c, d, e, and f are each preferably an integer of 1 to 30, more preferably an integer of 1 to 20. Z may be a combination of the above groups. In this case, c + d + e + f is preferably 1 to 40, more preferably about 1 to 20.
Therefore, when Z is composed of — (CH ₂ ) _c — alone, c is preferably an integer of 1 to 30, more preferably an integer of 1 to 20,
When Z is composed of — (CH ₂ —CH ₂ —O) _d — alone, d is preferably an integer of 1 to 30, more preferably an integer of 1 to 20,
When Z is constituted by-(Ph) _e -alone, e is preferably an integer of 1 to 30, more preferably an integer of 1 to 20,
When Z is composed of-(Ph-O) _f- alone, f is preferably an integer of 1 to 30, more preferably an integer of 1 to 20.

As described above, two or more kinds of the structural units described above may be included in any combination. Examples of such a configuration of Z include the following groups.
_{- (CH 2) c1 - (} Ph) e1 - (CH 2) c2 -
(C1 is an integer of 1 to 20, e1 is an integer of 1 to 20, c2 is an integer of 1 to 20, and c1 + e1 + c2 is 40 or less)
_{- (CH 2) c3 - (} Ph-O) f1 -
(C3 is an integer of 1 to 20, f1 is an integer of 1 to 20, and c3 + f1 is 40 or less)
_{- (CH 2) c4 - (} Ph-O) f2 - (CH 2 -CH 2 -O) d1 -
(C4 is an integer of 1 to 20, f2 is an integer of 1 to 20, d1 is an integer of 1 to 20, and c4 + f2 + d1 is 40 or less)
In the formula [13], a plurality of X ′, Y ′, and Z may be the same or different.

  Moreover, in Formula [14], a is an integer of 0-10, Preferably it is a product number of 0-3. Accordingly, a preferable cyclic sulfide moiety (SS bond-containing ring) in the formula [14] is represented by the following formula. In the following formula, z represents a bond that bonds to Z.

  JP, 2004-51624, A can be referred to for details, such as a synthesis method of the above-mentioned sulfur content compound. Such a sulfur-containing compound can be immobilized on a substrate having a metal surface by a known method. For example, the sulfur-containing compound can be immobilized on the substrate having a metal surface by immersing the substrate in a solution containing a sulfur-containing compound and allowing it to stand at room temperature for a predetermined time.

  In the present invention, the substrate on which the photodegradable protecting group-containing compound is immobilized can be appropriately selected according to the substance to be immobilized. Examples of the substrate include a glass substrate, a glass substrate on which a metal is deposited, a metal substrate, and a polystyrene substrate. As described above, when the photodegradable protective group-containing compound is an alkylsiloxane compound, it is preferable to use a glass substrate, and when the photodegradable protective group-containing compound is a sulfur-containing compound, it has a metal surface. It is preferable to use a substrate. As the substrate having a metal surface, a noble metal substrate such as a gold substrate, a silver substrate, or a platinum substrate, or a glass substrate on which a noble metal such as gold, silver, or platinum is deposited can be used. These substrates can be produced by a known method, and can also be obtained as commercial products.

Next, each aspect of the present invention will be described.

[First embodiment]
In the first aspect of the present invention, a compound having a terminal functional group protected by a photodegradable protecting group is immobilized on a substrate, and then at least a part of the photodegradable protecting group is eliminated by light irradiation to thereby terminate the terminal function. Expose at least part of the group. Then, the substrate on which the cells are immobilized is prepared by adsorbing the cells to at least a part of the exposed terminal functional group. Here, by partially irradiating light to remove a part of the photodegradable protecting group to expose the terminal functional group and adsorb cells on it, a substrate having a pattern in which cells are adsorbed is produced. can do. Furthermore, if a similar operation is performed again in other portions of the substrate after forming a kind of cell pattern to adsorb new cells, a substrate having a plurality of cell patterns of the same type or different types can be obtained.

The light used for elimination of the photodegradable protective group is not particularly limited as long as the photodegradable protective group can be eliminated and the terminal functional group can be exposed. For example, ultraviolet light (wavelength 1 to 400 nm) is used. Depending on the optical resolution of the photodegradable protecting group to be eliminated, light having a long wavelength (eg 365 nm) can be used as appropriate.
Light irradiation can be performed from the side of the substrate on which the photodegradable protective group-containing substance is immobilized, or from the opposite side. When performed from the opposite surface, the substrate is preferably made of a light-transmitting material such as glass. Moreover, if light irradiation is performed through the photomask in which the pattern was formed, the photodegradable protective group at a desired position can be selectively removed. The conditions for elimination of the photolabile protecting group vary depending on the type of the photodegradable protecting group and cannot be determined in general, but in general, a sufficient amount of ultraviolet light is irradiated at a temperature of about room temperature. By doing so, the photodegradable protecting group can be eliminated.

  The photodecomposition reaction of the photodegradable protecting group described above can be performed, for example, by using a mercury lamp provided in a fluorescence microscope as a light source. An example of a light irradiation system using a standard fluorescence microscope will be described with reference to FIG. First, as shown in FIG. 1, a photomask is inserted in a portion called a field stop. The photomask is provided with a pattern corresponding to the cell pattern to be formed on the substrate. This photomask can be produced, for example, by printing a desired pattern on an OHP sheet. Thereafter, the photodegradable protective group can be removed by selectively irradiating light having a desired wavelength corresponding to the photodegradable protective group through light from the mercury light source through an excitation filter.

  Thus, it is preferable to wash the substrate before and after exposing the terminal functional group by removing the photodegradable protecting group. The substrate can be washed using a known buffer.

  After washing the substrate as necessary, the cells are adsorbed on at least a part of the exposed terminal functional groups. Adsorption of cells can be performed by placing a substrate on which some terminal functional groups are exposed as described above in the presence of a medium containing cells and incubating for a predetermined time. Examples of terminal functional groups suitable for cell adhesion include amino groups based on the knowledge that cells (particularly nerve cells or cells into which genes have been introduced) are likely to adhere to a positively charged surface. However, since the cell may adhere to a negatively charged surface or a neutral surface, the type of the terminal functional group is preferably selected according to the cell.

  In the first aspect, in order to suppress the adsorption of cells to the substrate surface other than the portion where the terminal functional group is exposed, the photodegradable protective group preferably has no adsorptivity to the cells to be used. . Thereby, even in the presence of cells over the entire substrate, cells can be selectively adsorbed only to the terminal functional group. Since cells tend not to adhere to a hydrophilic surface, cell adhesion can be effectively suppressed if the photodegradable protective group is highly hydrophilic. In addition, when a neutral compound having a group having a hydrogen bond acceptor is used as the photodegradable protective group-containing compound, cell adsorption to the substrate surface other than the portion where the terminal functional group is exposed can be effectively suppressed. There is.

  The cells used in the first, second, and third aspects of the present invention are primary cultured cells derived from nerves, endothelium, skin, lungs, muscles, kidneys, liver, intestines, etc., and their cancerous cells and cells. Strains (HepG2, HUVEC, CHO, PC12, HeLa, MDCK, etc.). In addition, somatic / embryonic stem cells as well as recombinant somatic cells into which genes have been introduced can also be used. Furthermore, cells other than mammalian cells can be used, and their biological species are not limited. In the present invention, for example, HEK293 cells, COS7 cells, and NIH3T3 cells can be used.

The number of cells added to the medium is not particularly limited as long as the adhesion region is sufficiently large. Practically, it is appropriate to add a sufficiently large number of cells so that the cell and the adhesion region can collide in about 2 hours. For example, when forming a cell adhesion region of about 0.25 mm ^{2 with} respect to a 3.5 cm diameter dish, it is sufficient to add 10 ⁵ order cells. Moreover, the culture medium to be used can be suitably selected from the culture media suitable for culture | cultivation of each cell, and a well-known culture medium can be used. When it is desired to keep cells strictly in the adhesion region, it is preferable to use a medium that does not contain serum at the time of cell seeding, and replace the medium with serum after 1 to 2 hours.

  After the above operation, light irradiation is performed using a photomask formed with another pattern, and a part of the photodegradable protecting group that has not been removed by the previous light irradiation is removed to expose the terminal functional group, A new cell pattern can be formed by adsorbing at least a part of the exposed terminal functional group to cells of the same or different type as the previously adsorbed cells. A new cell pattern is formed by replacing the medium containing the substrate with a medium not containing cells, then irradiating with light using a photomask corresponding to the new pattern, and then adding new cells to the medium. It can be carried out. At this time, if a different type of cell from the previously adsorbed cells is adsorbed, a substrate having two types of cell patterns can be formed. Further, if the same operation is performed, it is possible to form three or more types of cell patterns.

[Second embodiment]
In the second aspect of the present invention, after immobilizing the photodegradable protective group-containing compound on the substrate, the cell adhesion inhibitor is adsorbed on at least a part of the photodegradable protective group. Adsorption of the cell adhesion-inhibiting substance can be performed by immersing the substrate in a solution containing the cell adhesion-inhibiting substance or by dropping a solution containing the cell adhesion-inhibiting substance on the substrate and leaving it for a predetermined time. Prior to adsorption of the cell adhesion-inhibiting substance, the substrate can be sterilized with, for example, ethanol. Moreover, it is preferable to wash the substrate before and after adsorbing the cell adhesion inhibitor. The substrate can be washed using a known buffer.

  Examples of the cell adhesion inhibitor include proteins (serum albumin, ovalbumin, casein, kininogen, etc.), polymers (polyethylene glycol, 2-methacryloyloxyethyl phosphorylcholine (MPC), agarose, polysaccharides, and derivatives thereof), low Molecular compounds (oligoethylene glycol, mannitol, and derivatives thereof, natural / synthetic phospholipids (phosphatidylcholine, etc.)) can be used, and it is preferable to select them according to the cells to be used. Among them, it is preferable to use serum albumin because it has an adhesion suppressing effect on many cells. In addition, the cell adhesion inhibitory substances exemplified here can be applied to many cell types, but there are also cells that secrete proteases that digest cell adhesion inhibitory proteins. Alternatively, phospholipid is preferably used. In selecting a cell adhesion inhibitor, it is preferable to consider the adsorptivity to the photodegradable protecting group.

  A solution containing a cell adhesion inhibitory substance can be prepared by dissolving the cell adhesion inhibitory substance in an appropriate buffer. The concentration of the cell adhesion-inhibiting substance in the solution is preferably adjusted as appropriate according to the cell adhesion-inhibiting effect of the cell adhesion-inhibiting substance used, and can be, for example, 1 μg / ml to 20 mg / ml. As the buffer, a buffer having a buffer capacity near neutrality can be used. For example, a phosphate buffer, a carbonate buffer, a HEPES buffer, or the like can be used. Further, the concentration of the buffer is preferably in a concentration range in which the buffering capacity is exerted, and is such a concentration that does not lose the activity of the cell adhesion inhibitory substance. For example, 100 to 200 mM is practical.

  Thereafter, at least a part of the photodegradable protective group to which the cell adhesion inhibitor is adsorbed by light irradiation is removed to expose at least a part of the terminal functional group. The elimination of the photodegradable protecting group by this light irradiation is as described in the first embodiment, and the terminal functional group is exposed at a desired position on the substrate by performing partial light irradiation. Can be made. By adsorbing the cells to the terminal functional groups thus exposed, a substrate on which the cells are immobilized can be obtained. The cell adsorption method is as described in the first embodiment. In the second aspect, since the cell adhesion inhibitor is adsorbed on the substrate surface other than the portion where the terminal functional group is exposed, cell adhesion is suppressed. Therefore, there is an advantage that cells can be selectively adsorbed only to the terminal functional group even in the presence of cells over the entire substrate.

  Also in the second embodiment, the photodegradable protective group (cell adhesion inhibitory substance adsorbed) that has not been detached by the previous light irradiation after light irradiation using a photomask formed with another pattern A new cell pattern can be formed by removing a part to expose the terminal functional group and adsorbing the same or different cells as the cells previously adsorbed to the exposed terminal functional group. For the formation of a new cell pattern, the medium containing the substrate is replaced with a medium that does not contain cells, and then light irradiation is performed using a photomask corresponding to the new pattern, and then new cells are added to the medium. Can be done by. At this time, if a different type of cell from the previously adsorbed cells is adsorbed, a substrate having two types of cell patterns can be formed. Further, if the same operation is performed, it is possible to form three or more types of cell patterns.

[Third embodiment]
In the third aspect of the present invention, a cell adhesion inhibitory substance is adsorbed on at least a part of the photodegradable protective group, and at least a part of the photodegradable protective group adsorbed by the cell adhesion inhibitory substance is removed by light irradiation. To expose at least a portion of the terminal functional group. About these operation, it is as having described the 2nd aspect previously, A terminal functional group can be exposed to the desired position on a board | substrate by performing light irradiation partially. Thereafter, the cell adhesion promoting substance is adsorbed on at least a part of the exposed terminal functional group. Adsorption of the cell adhesion promoting substance can be performed by immersing the substrate in a solution containing the cell adhesion promoting substance or by dropping a solution containing the cell adhesion promoting substance on the substrate and leaving it for a predetermined time. It is preferable to wash the substrate before and after adsorbing the cell adhesion promoting substance. The substrate can be washed using a known buffer.

  Examples of cell adhesion promoters include proteins (fibronectin, collagen, laminin, tenascin, etc.), polymers (polylysine, RGD peptide modified polymers, etc.), low molecular compounds (adhesion protein mimetic peptides (RGD peptide, PHSRN peptide), etc.) ) Can be used and is preferably selected according to the cell used. Among these, fibronectin is preferable because it has cell adhesion to many cells. As the cell adhesion promoting substance, it is preferable to use a substance (protein, its mimetic substance and fragment thereof) that becomes a ligand for the receptor protein expressed in the cell membrane of the cell type to be used. For example, laminin is effective for nerve cells, and collagen is effective for endothelial cells, hepatocytes, muscle cells, and the like. In addition, for nerve cells and cells into which genes have been introduced, a positively charged polymer compound such as polylysine is also effective. In selecting a cell adhesion promoting substance, it is preferable to consider the adsorptivity to the terminal functional group.

  A solution containing a cell adhesion promoting substance can be prepared by dissolving the cell adhesion promoting substance in an appropriate buffer. The concentration of the cell adhesion promoting substance in the solution is preferably adjusted as appropriate according to the cell adhesion property of the cell adhesion promoting substance to be used, and can be, for example, 1 μg / ml to 20 mg / ml. As the buffer, a buffer having a buffering ability near neutrality can be used. For example, a phosphate buffer, a carbonate buffer, a HEPES buffer, or the like can be used. Further, the concentration of the buffer is preferably in a concentration range in which the buffering capacity is exerted, and is such a concentration that the activity of the cell adhesion promoting substance is not lost. For example, 100 to 200 mM is practical.

  After the cell adhesion promoting substance is adsorbed, a cell on which the cells are immobilized can be obtained by adsorbing the cells to at least a part of the cell adhesion promoting substance. Cell adsorption can be performed in the same manner as described above for the first embodiment. In particular, in the third aspect, since the cells are adsorbed to the cell adhesion promoting substance, there is an advantage that the cell is favorably adsorbed as compared with the methods of the first and second aspects. Further, in the third aspect, similarly to the second aspect, since the cell adhesion inhibitor is adsorbed on the substrate surface other than the portion where the terminal functional group is exposed, cell adhesion is suppressed. Therefore, there is an advantage that cells can be selectively adhered to a cell adhesion promoting substance on the substrate even in the presence of cells.

  Also in the third aspect, photoirradiation using a photomask having another pattern is performed, and a photodegradable protective group (cell adhesion inhibitor is adsorbed) that has not been detached by the previous light irradiation. A part of the terminal functional group is removed to expose the terminal functional group, and a cell adhesion promoting substance that is the same or different from the cell adhesion promoting substance previously adsorbed to at least a part of the exposed terminal functional group is adsorbed to the cell. A new cell pattern can be formed by adsorbing at least a part of the cells of the same or different type as the previously adsorbed cells. The new cell pattern is formed by irradiating light using a photomask corresponding to the new pattern, replacing the medium containing the substrate with the medium containing the cell adhesion promoting substance before or after the light irradiation, and then newly Can be performed by adding various cells to the medium. At this time, if a different type of cell from the previously adsorbed cells is adsorbed, a substrate having two types of cell patterns can be formed. Further, if the same operation is performed, it is possible to form three or more types of cell patterns.

  According to the method of the 1st, 2nd, 3rd aspect of this invention demonstrated above, formation of a new cell adhesion pattern and a change of size can be performed after cell seeding. In particular, elimination of the photodegradable protective group, adsorption of cells, cell adhesion-inhibiting substances, and cell adhesion promoting substances in the method of the present invention can be easily performed even in cell culture. Therefore, according to the method of the present invention, it is possible to easily form a new cell adhesion pattern and change the size under cell culture.

  The substrate on which the cells obtained by the method of the present invention are immobilized can be a cell array. The cell array refers to a substrate on which spots on which cells are immobilized are formed in an array. According to the method of the present invention, cells having a desired cell pattern are obtained by irradiating light through a photomask having a pattern and selectively desorbing a photodegradable protecting group at a desired position on the substrate. Arrays can be made easily.

  Furthermore, the present invention is a substrate in which a compound having a terminal functional group protected by a photodegradable protecting group is immobilized on a substrate, and a cell adhesion inhibitor is adsorbed on at least a part of the photodegradable protecting group. It also relates to the substrate. The method for manufacturing the substrate of the present invention is as described above. The substrate of the present invention can be used in the second and third aspects of the present invention.

Hereinafter, the present invention will be described with reference to examples.

[Example 1, comparative example]
(1) Immobilization of a photodegradable protecting group-containing compound on a substrate
According to Chem. Lett. 29, 228-229 (2000), 5-trichlorosilylpentanoic acid (2-nitrophenyl) ethyl ester (TCS-PNPE) was synthesized.
A cover glass (manufactured by Matsunami) was heated in a mixed solution of H ₂ SO ₄ : H ₂ O ₂ at 90 to 100 ° C. for 1.5 hours. Subsequently, ultrasonic treatment was performed after washing with pure water. A cover glass was placed in a benzene solution (12 mM) of TCS-PNPE, refluxed for 1 hour, and allowed to stand at room temperature for 5 minutes. After washing with methanol and chloroform, ultrasonic treatment was performed in chloroform, and the surface was dried with a nitrogen stream to obtain a cover glass on which TCS-PNPE was immobilized.

(2) Protein surface adsorption and cell seeding
Place a piece of cover glass on which TCS-PNPE is immobilized on the bottom of a glass bottom dish (MatTek), sterilize with ethanol, and in PBS solution of 10 mg / ml bovine serum albumin (BSA, Wako Pure Chemical Industries) (Dulbecco) PBS (manufactured by Wako Pure Chemical Industries, Ltd.) was allowed to stand at room temperature for 1 hour to adsorb BSA onto the substrate surface. After washing with PBS, the sample was irradiated with light using an excitation filter (330 ± 40 nm) and a 10x objective lens (Plan Apochromat, Zeiss) under the fluorescence microscope Axiovert 200 (supplied with 103W mercury light source) manufactured by Carl Zeiss (150 A Seconds). At that time, a photomask in which the required cell pattern was printed on the OHP sheet was inserted into the position of the field stop slider of the microscope, and patterned irradiation was performed. After irradiation with light, the plate was again washed with PBS, subsequently replaced with a PBS solution containing 25 μg / ml fibronectin (BD), and incubated at room temperature for 30 minutes. After washing with PBS again, the buffer was replaced with MEM medium without serum, 4-8 x 10 ⁵ HEK293 cells (manufactured by JCRB) were seeded, and the cells were allowed to adhere for 2 hours in a CO ₂ incubator. The normal medium containing was replaced. The same operation was performed using COS7 cells and NIH3T3 cells (manufactured by RIKEN BRC). Furthermore, the same operation was performed on HEK293 cells using an organosiloxane (2-trichlorosilylacetic acid methyl ester) having no photodegradable protecting group (Comparative Example). Cells were observed by taking a phase contrast image using a 10 × or 20 × phase contrast objective lens (Plan Neofluar, Zeiss) using a fluorescence microscope. 2A shows a light irradiation region, FIG. 2B shows a phase contrast photograph 2 hours after HEK293 cell seeding, and FIG. 2C shows a phase contrast photograph 63 hours after HEK293 cell seeding. 2 (d) shows the results of a comparative example using an alkylsiloxane having no photodegradable protecting group, FIG. 2 (e) shows 18 hours after COS7 cell seeding, and FIG. 2 (f) shows NIH3T3 cell seeding. A phase contrast photograph after 18 hours is shown.

(3) Results of light switching of cell adhesion From FIG. 2, HEK293 cells selectively adhere to the light irradiation region corresponding to the circular light irradiation region (FIG. 2 (a)) (FIG. 2 (b)). And stayed within the irradiated area for about 3 days to grow (FIG. 2 (c)). In addition to HEK293 cells, the same phenomenon was observed in COS7 cells and NIH3T3 cells (FIGS. 2 (e) and (f)). On the other hand, such switching of cell adhesion by light irradiation was not observed in organosiloxane (2-trichlorosilylacetic acid methyl ester) having no photodegradable protecting group (FIG. 2 (d)).

  Moreover, the result of having analyzed the surface adsorption | suction protein in the process of light irradiation with the immuno-staining method is shown to FIG.2 (g), (h). In the light irradiation region, BSA was first desorbed (FIG. 2 (g)), and then fibronectin was adsorbed (FIG. 2 (h)). Thereby, in the method of this invention, it turned out that the exchange of BSA and a fibronectin occurs selectively in the light irradiation area | region, and has promoted the adhesion of a cell. Since fibronectin is known to promote the adhesion of many cell types, the method of the present invention shows that the pattern of fibronectin adhesion is formed by light irradiation in the method of the present invention. This means that the method is generally applicable to other than the three types of cells.

[Example 2]
Various cell adhesion pattern formation In order to obtain various patterns, cell adhesion regions were formed using a photomask on which spots of different size arrays were printed. FIGS. 3A, 3B, and 3D show HEK293 cell seeding on a substrate prepared by inserting a photomask on which a desired pattern is printed into a field stop of a fluorescence microscope and using the same method as in Example 1. FIG. FIG. 3 (c) shows a phase contrast photograph after a day, and a photomask printed with a desired pattern is inserted into a field stop of a fluorescence microscope, and a substrate prepared in the same manner as in Example 1 is obtained after 2 hours of HEK293 cell seeding. It is a photograph fixed with paraformaldehyde. The arrow in FIG.3 (c) shows the nodal structure by the adhesion spot of the cell having been formed.
As shown in FIG. 3, the cell spot of multi-cell (FIG. 3 (a), 120 μm), single cell (FIG. 3 (b), 30 μm), subcell (FIG. 3 (c), 6 μm) size is obtained by the method of the present invention. In addition, a cell adhesion region having a stripe shape (FIG. 3D, 60 μm) could be formed. From the above results, according to the method of the present invention, it is possible to form a cell adhesion region of any size and position very easily, and the spatial resolution thereof is smaller than the cell size (subcell) level. I found out that

[Example 3]
Positioning of another cell in the vicinity thereof in the culture environment of the previously adhered cell By the method described in Example 1, a substrate on which HEK293 cells (unstained) were adsorbed was prepared and cultured overnight. Thereafter, in order to adsorb new cells in the vicinity of the previously adsorbed cells, light irradiation was performed using a photomask formed with a pattern (square area in FIG. 4A). Thereafter, the medium was replaced with a normal medium supplemented with 25 μg / ml fibronectin, and then 4-8 × 10 ⁵ HEK293 cells previously stained with a fluorescent dye Cell Tracker Green CMFDA (Molecular Probes) were seeded. Cells were adsorbed for 3 hours in a CO ₂ incubator and then replaced with normal medium. Take a phase contrast image using a 10x or 20x phase contrast objective lens (Plan Neofluar, Zeiss) using a fluorescence microscope, observe unstained cells, and view fluorescence images of cells stained with Cell Tracker. Images were taken using an excitation filter (475 ± 10 nm), a fluorescence filter (545 ± 18 nm) and a dichroic mirror (500DRLP). The results are shown in FIG. FIG. 4 (a) is a phase contrast image showing a cell pattern of unstained HEK293 cells, FIG. 4 (b) is a phase contrast photograph 14 hours after adsorbing HEK293 cells, and FIG. 4 (c) is It is the fluorescence photograph. As shown in FIGS. 4B and 4C, according to the method of the present invention, it was observed that cells selectively adhere to the light irradiation region. Thus, according to the method of the present invention, another cell can be positioned in the immediate vicinity of the previously adhered cell in a cell culture environment.

  According to the present invention, the cell adhesion region can be freely designed and can be formed under a standard fluorescence microscope. As described above, according to the method of the present invention, it is possible to co-culture a plurality of types of cells on one substrate by sequentially forming cell adhesion regions. The method of the present invention is useful as a research tool for the interaction between allogeneic / heterologous cells, and is a user-friendly method that can be easily performed under a standard fluorescence microscope.

An example of the light irradiation system using a standard fluorescence microscope is shown. The result of Example 1 and a comparative example is shown. The result of Example 2 is shown. The result of Example 3 is shown.

Claims (10)

A method for producing a substrate on which cells are immobilized,
Immobilizing a compound whose terminal functional group is protected by a photodegradable protecting group on a substrate,
Removing at least part of the photodegradable protecting group by light irradiation to expose at least part of the terminal functional group;
The method, wherein cells are adsorbed to at least a part of the exposed terminal functional group. After the cell adsorption, at least part of the photodegradable protecting group that does not desorb by light irradiation is desorbed by light irradiation to expose at least part of the terminal functional group,
The method according to claim 1, wherein a cell that is the same or different from the cell is adsorbed to at least a part of the exposed terminal functional group. A method for producing a substrate on which cells are immobilized,
Immobilizing a compound whose terminal functional group is protected by a photodegradable protecting group on a substrate,
Adsorbing a cell adhesion inhibitor on at least a part of the photodegradable protecting group;
Removing at least part of the photodegradable protecting group adsorbed by the cell adhesion inhibitor by light irradiation to expose at least part of the terminal functional group;
The method, wherein cells are adsorbed to at least a part of the exposed terminal functional group. After the cell adsorption, at least a part of the photodegradable protective group adsorbed by the cell adhesion inhibitor that does not desorb by the light irradiation is desorbed by the light irradiation to expose at least a part of the terminal functional group,
The method according to claim 3, wherein a cell that is the same or different from the cell is adsorbed to at least a part of the exposed terminal functional group. A method for producing a substrate on which cells are immobilized,
Immobilizing a compound whose terminal functional group is protected by a photodegradable protecting group on a substrate,
Adsorbing a cell adhesion inhibitor on at least a part of the photodegradable protecting group;
Removing at least part of the photodegradable protecting group adsorbed by the cell adhesion inhibitor by light irradiation to expose at least part of the terminal functional group;
Adsorbing a cell adhesion promoting substance on at least a part of the exposed terminal functional group;
The method described above, wherein cells are adsorbed to at least a part of the cell adhesion promoting substance. After the cell adsorption, at least a part of the photodegradable protective group adsorbed by the cell adhesion inhibitor that does not desorb by the light irradiation is desorbed by the light irradiation to expose at least a part of the terminal functional group,
Adsorbing a cell adhesion promoting substance that is the same or different from the cell adhesion promoting substance on at least a part of the exposed terminal functional group;
The method according to claim 5, wherein cells that are the same or different from the cells are adsorbed to at least a part of the cell adhesion promoting substance. The method according to claim 1, wherein the light irradiation is performed through a photomask. The method according to claim 1, wherein the terminal functional group is a carboxyl group. The method according to claim 1, wherein the substrate on which the cells are immobilized is a cell array. A substrate on which a compound having a terminal functional group protected by a photodegradable protecting group is immobilized on a substrate, wherein a cell adhesion inhibitor is adsorbed on at least a part of the photodegradable protecting group .