WO2019230441A1

WO2019230441A1 - Photoresponsive cell fixing agent

Info

Publication number: WO2019230441A1
Application number: PCT/JP2019/019632
Authority: WO
Inventors: 哲志山口; 森泉田; 岡本　晃充
Original assignee: 国立大学法人東京大学
Priority date: 2018-05-31
Filing date: 2019-05-17
Publication date: 2019-12-05
Also published as: JP2019208381A; JP7236126B2

Abstract

[Problem] The present invention addresses the problem of developing a photoresponsive cell fixing agent that can be used repeatedly and continuously, and can selectively and reversibly control the fixing and releasing of cells by photostimulation, regardless of the targeted cell type. [Solution] A photoresponsive cell fixing agent for fixing prescribed target cells on a substrate, said photoresponsive cell fixing agent being characterized by having a hydrophobic chain that can interact with the target cells, a hydrophilic chain that can be arranged in a monomolecular film shape on a surface of the substrate, and a branched linker chain that links the hydrophobic chain and the hydrophilic chain, wherein the photoresponsive cell fixing agent has, on a side chain of the branched linker chain, a photoisomerization site that is isomerized by light irradiation and can change from hydrophobic to hydrophilic.

Description

Photoresponsive cell fixing agent

The present invention relates to a photoresponsive cell fixing agent having excellent cell fixing power, a cell immobilization substrate having a surface modified with the photoresponsive cell fixing agent, and a cell recovery method using the substrate. .

In recent years, techniques for manipulating specific cells one by one have attracted a great deal of attention with the development of regenerative medicine and single cell analysis. As a technique for manipulating such a single cell, for example, a mechanical method using a micromanipulator or the like is widely used, but it has a drawback that a large number of cells cannot be handled at one time. Therefore, the development of a substrate having a stimulus-responsive surface that can selectively immobilize and detach cells by external stimulation is expected as a technique that can manipulate cells with high throughput.

Typically, in such a technique using a stimulus-responsive substrate, cells can be fixed and arranged at a desired location by stimulation, and cell processing such as phenotype and other cell analysis and gene transfer can be performed. After that, only the target cell location is stimulated exhaustively, so that only the cells with the target phenotype or the target processed cells are selectively detached at the single cell level and selected. Is possible.

Furthermore, as an advantage of the technique using a substrate having a stimulus-responsive surface, it can be overwhelmingly simple to perform parallel processing of cells simultaneously as compared with a conventional mechanical technique. . Moreover, by incorporating such a substrate surface into the device, it is expected that high-speed and large-scale automatic preparation of cells for regenerative medicine will be possible, or quick and comprehensive automatic single-cell diagnosis will be possible. Therefore, there is a need for the development of a substrate surface that can repeatedly fix and detach individual cells in response to external stimuli within the device.

Substrate surfaces and substrate surface preparation techniques that can selectively fix and detach cells by external stimulation can be broadly classified into the following four types depending on the type of stimulation used: (1) Technology using photoresponsive materials, (2) Technology using a thermally responsive material, (3) Technology using an electrically responsive material, (4) Technology using a material that responds to other stimuli such as addition of chemicals and biomolecules. Here, all of the external stimuli used in (2) to (4) have low spatial resolution, and it is difficult to give stimuli only to a single cell, and therefore cannot be used for operation at the single cell level. . On the other hand, light, which is an external stimulus used in (1), has an advantage that it has a high spatial resolution and can irradiate only a single cell. Therefore, an application to a technique for producing a light-responsive substrate surface that can be manipulated at the level of one cell as described above and that allows repeated cell immobilization has been attempted. However, the existing technique using light stimulation can be applied only to adherent cells, and there is a problem that it cannot handle suspended cells and suspended cells, which are the main targets of cell manipulation.

On the other hand, in the research group of the present inventors, any cell can be manipulated regardless of cell adhesion by using a compound in which polyethylene glycol (PEG) and lipid are linked via a photodegradable linker. A photoinactive type and a photoactive type cell fixing agent have been reported (Non-patent Document 1). However, the cell immobilization agent can be used only once for immobilization or detachment by light stimulation, and it is not possible to operate repeatedly by switching between immobilization and detachment of cells. It wasn't.

In view of the problems in the prior art, the present invention can selectively and reversibly control cell immobilization and detachment by light stimulation regardless of the target cell type, and can be used repeatedly and continuously. It is an object of the present invention to develop a photoresponsive cell fixing agent that can be used.

As a result of intensive studies to solve the above problems, the present inventor has made a hydrophobic / hydrophilic hydrophilic group by photoisomerization as a side chain to a linker site that connects a hydrophobic site capable of binding to a target cell and a hydrophilic site. By using a molecule having a branched linker into which a structure that can change is used, by selectively irradiating light of a specific wavelength, arbitrary cells are selectively fixed on the substrate and irradiated with light of another wavelength. Thus, it has been found that the target cells can be repeatedly fixed and detached by alternately irradiating light of two wavelengths, and the present invention has been completed.

That is, the present invention in one aspect,
<1> A photoresponsive cell immobilizing agent for immobilizing a predetermined target cell on a substrate, a hydrophobic chain capable of interacting with the target cell, and a monomolecular film on the surface of the substrate It has a hydrophilic chain that can be arranged, and a branched linker chain that connects the hydrophobic chain and the hydrophilic chain. The side chain of the branched linker chain is isomerized by light irradiation and changes from hydrophobic to hydrophilic. A photoresponsive cell fixing agent characterized by having a photoisomerization site capable of
<2> The amount of change in the logarithmic value (logP) of the distribution coefficient due to the isomerization of the photoisomerization site is 0.5 or more (where the distribution coefficient includes the hydrophobic chain (a) and the branched linker) A calculated value of the partition coefficient in the region consisting of the chain (c)), the photoresponsive cell fixing agent according to <1>above;
<3> The photoresponsive cell fixing agent according to the above <2>, wherein a logarithmic value (logP) of a partition coefficient in a more hydrophobic isomer before and after isomerization of the photoisomerization site is 10 or more. ;
<4> The photoresponsive cell fixing agent according to <1>, wherein the photoisomerization site includes a structure of spiropyran or a derivative thereof;
<5> The photoresponsive cell fixing agent according to <1>, wherein the photoisomerization site has a structure represented by the following formula (I):

(Wherein R ₁ is a hydrogen atom or an alkyl group; R ₂ is a C ₁ to C ₂₀ alkylene group; R _A , R _B , and R _C may be the same or different; Independently a hydrogen atom, an alkyl group, an alkoxy group, or a nitro group; * is a binding moiety to the branched linker chain);
<6> The photoresponsive cell fixing agent according to <5>, wherein R ₂ is a C ₅ or C ₆ linear alkylene group; and R _C is a nitro group;
<7> The photoresponsive cell fixing agent according to any one of the above <1> to <6>, wherein the hydrophobic chain is a saturated or unsaturated hydrocarbon chain which may have a substituent. ;
<8> The photoresponsive cell fixing agent according to any one of <1> to <7>, wherein the hydrophilic chain includes a hydrophilic polymer;
<9> The photoresponsive cell fixing agent according to <8>, wherein the hydrophilic polymer is polyalkylene glycol;
<10> The photoresponsive cell fixing agent according to any one of the above <1> to <9>, wherein the branched linker chain has an amino acid residue or a repeating structure thereof;
<11> The photoresponsive cell fixing agent according to any one of <1> to <10>, wherein the photoisomerization site is linked to the branched linker chain through an amide bond;
<12> The photoresponsive cell fixing agent according to any one of the above <1> to <11>, which has a substituent capable of being covalently bonded to the surface of the base material at the end of the hydrophilic chain; And <13> the photoresponsive cell fixing agent according to <1>, which has the following structure:

(In the formula, Sp represents the photoisomerization site; m is a natural number of 1 to 5; and n is a natural number of 50 to 500.)
Is to provide.

In another aspect, the present invention provides:
<14> A cell immobilization substrate having a surface modified with the photoresponsive cell immobilizing agent according to any one of <1> to <13>.

In a further aspect, the invention provides:
<15> (i) irradiating the cell immobilization substrate according to <14> above with light of a specific wavelength, and converting the photoisomerization site into a hydrophilic isomer structure;
(Ii) a step of bringing a solution containing predetermined target cells into contact with the substrate for cell immobilization, and immobilizing the target cells on the substrate for cell immobilization; and (iii) the substrate for cell immobilization. Is irradiated with light having a wavelength different from that in the step (i), and the photoisomerization site is converted into a hydrophobic isomer structure, whereby the immobilized target cells are removed from the cell immobilization substrate. Separation / recovery process,
A cell recovery method comprising:
<16> The method for recovering cells according to the above <15>, comprising repeating the steps (i) to (iii) a plurality of times;
<17> The cell collection according to <15> or <16>, wherein the light irradiated in the step (i) is ultraviolet light; and the light irradiated in the step (iii) is visible light A method is provided.

According to the photoresponsive cell immobilizing agent of the present invention, by reversibly changing the hydrophobicity and hydrophilicity by the structural change of the photoisomerization site by light irradiation, it is possible to stimulate light regardless of the target cell type. Can be selectively controlled at the single cell level. Then, the optical responsive cell immobilization / detachment process can be repeatedly and continuously performed by optical switching in which two wavelengths of light are alternately irradiated.

Since the present invention is a new and powerful method for quickly and accurately selecting only the target cells from a large number of cells, it can be used in basic research in the field of cell biology, antibody selection in pharmaceutical companies, and discovery research for drug discovery targets. Can be expected to be widely applied.

FIG. 1 is a schematic diagram showing the overall structure of the photoresponsive cell fixing agent of the present invention. FIG. 2 is a graph showing the cell immobilization density accompanying light irradiation for the substrate modified with the photoresponsive cell fixing agent of the present invention. (A) Compound 1, (b) Compound 1b, (c) Compound 1c, (d) Compound 1d, and (e) Compound 1e. FIG. 3 is a graph showing changes in cell immobilization density when a substrate modified with the photoresponsive cell immobilization agent (compound 1d) of the present invention is irradiated with visible light after cell immobilization. FIG. 4 is a graph showing the number of cell immobilization during the repeated operations of cell immobilization and detachment by light irradiation for a substrate modified with the photoresponsive cell immobilizing agent (compound 1d) of the present invention. FIG. 5 is a fluorescence microscopic image of cells on the surface irradiated with ultraviolet light and visible light, respectively, on a substrate modified with the photoresponsive cell fixing agent (compound 1d) of the present invention. FIG. 6 shows the number of cell immobilizations when various operations are performed on cells modified with a light-responsive cell immobilization agent (compound 1d) of the present invention by repeatedly repeating cell immobilization and detachment by light irradiation. (Off: after irradiation with visible light, on: after irradiation with ultraviolet light). (A) K562 cells, (b) Jurkat cells, (c) HeLa cells. FIG. 7 shows the same time as the survival rate (Released) of the cells immobilized on the substrate modified with the photoresponsive cell fixing agent (compound 1d) of the present invention and then removed by irradiation with visible light. It is a graph which shows the survival rate (Pos. Ctrl.) Of a control cell in PBS.

Hereinafter, embodiments of the present invention will be described. The scope of the present invention is not limited to these descriptions, and other than the following examples, the scope of the present invention can be appropriately changed and implemented without departing from the spirit of the present invention.

1. Photoresponsive cell fixing agent The photoresponsive cell fixing agent of the present invention comprises:
(A) a hydrophobic chain having a function of interacting with a target cell and binding to the cell;
(B) a hydrophilic chain that can be arranged in the form of a monomolecular film on the surface of the substrate, and (c) a branch having a branched structure with a side chain as a linker for connecting the hydrophobic chain and the hydrophilic chain. And a photoisomerization site (d) which can be isomerized by irradiation with light and change from hydrophobic to hydrophilic in the side chain of the branched linker chain.

FIG. 1 shows the entire structure of the photoresponsive cell fixing agent of the present invention. For the connection of each site, for example, a covalent bond such as an amide bond, an ester bond, an ether bond, a thioether bond, a carbamate bond, a thiocarbamate bond, a triazole bond, or a urea bond can be used. In addition, it is preferable that the photoisomerization site (side chain) in the branched linker chain (c) is connected to the branched linker chain (its main chain) by an amide bond.

The photoresponsive cell fixing agent of the present invention typically modifies the substrate surface by binding to the substrate surface directly or via a coating layer described later at the end of the hydrophilic chain (b). Used. In this case, the photoresponsive cell fixing agent is preferably arranged in the form of a monolayer on the surface of the substrate. On the other hand, the hydrophobic chain (a) can be bound to and captured by the target cell by an interaction such as a hydrophobic interaction. Thereby, a target cell can be fixed to the specific area | region of the base-material surface. In addition, by irradiating a desired region on the surface of the substrate with light of a specific wavelength, the photoisomerization site (d) in the side chain of the branched linker chain (c) is structurally changed by photoisomerization, and its hydrophobicity By changing, the target cells can be selectively separated from the surface of the substrate and recovered. Furthermore, by irradiating the substrate surface with light of a specific wavelength again, the photoisomerization site (d) can be returned to the original structure by re-photoisomerization, and cell immobilization and separation / recovery can be performed. It becomes possible to carry out repeatedly.

Here, the “cell” can include an animal cell, a plant cell, an insect cell, a prokaryotic cell, a fungal cell, and the like, and generally does not adhere or extend to the surface of a carrier such as a culture instrument, and is in a suspended or precipitated state. Proliferating “floating cells” (eg, blood cells) and “adherent cells” that adhere to and spread on the carrier surface are dispersed from the carrier with an appropriate dispersant such as EDTA-trypsin or dispase, and suspended temporarily. (For example, fibroblasts detached from the carrier with EDTA solution) and cells adhered to the carrier. Also included are living organisms having a phospholipid bilayer on the surface, such as liposomes, exosomes, bacteria, viruses, organelles, plant cells from which the cell walls have been removed (protoplasts). In addition, according to the photoresponsive cell fixing agent of the present invention, a substance having lipid such as lipid-coated particles can also be immobilized.

The hydrophobic chain (a) in the photoresponsive cell fixing agent of the present invention is a site for binding with target cells and capturing the target cells. As such an interaction, a non-covalent interaction such as a hydrophobic interaction can be used. Specifically, the hydrophobic chain (a) can bind to a target cell by a hydrophobic interaction with a lipid moiety in a cell membrane or the like that is a lipid bilayer membrane.

The hydrophobic chain (a) is not particularly limited as long as it can bind to the target cell by hydrophobic interaction, but may be a saturated or unsaturated hydrocarbon chain which may have a substituent. Examples of such hydrocarbon chains include, for example, C _7-30 alkyl groups (preferably C _7-22 alkyl groups), C _6-14 aryl groups, C _6-14 aryl C _7-30 alkyl groups (preferably C ₇ _6-14 aryl C _7-22 alkyl group) and C _7-30 alkyl C _6-14 aryl group (preferably C _6-14 aryl C _7-22 alkyl group). Preferably, adjacent carbon atoms may be connected by 1 to 3 unsaturated bonds, C _7-30 alkyl group, and adjacent carbon atoms may be connected by 1 to 3 unsaturated bonds. A C _7-22 alkyl group, or a C _11-22 alkyl group in which adjacent carbon atoms may be linked by 1 to 3 unsaturated bonds, or 1 to 3 unsaturated bonds in adjacent carbon atoms It may be a C _16-18 alkyl group which may be linked. More preferably, the hydrophobic chain (a) is a hexadecyl group, a heptadecyl group, an octadecyl (stearyl) group, a cis-9-hexadecenyl (palmitoleyl) group, a cis-8-heptadecenyl group, a trans-8-heptadecenyl group, a trans -9-octadecenyl (elaidyl) group, cis-9-octadecenyl (oleyl) group, cis, cis-9,12-octadecadienyl (linolenyl) group, (9E, 12E, 15E) -octadec-9,12 It can be a 15-trienyl (eridolinolenyl) group. In particular, an oleyl group that is a part of the phospholipid constituting the cell membrane is preferable. Furthermore, these hydrophobic chains may be substituted with an arbitrary substituent, and may contain heteroatoms such as N, S, and O.

The hydrophilic chain (b) is preferably composed of a hydrophilic polymer. Examples of such hydrophilic polymers include polyalkylene glycol, polyvinyl alcohol, polyacrylic acid, polypeptides, polyacrylamide, and polysaccharides such as dextran, or polymers and copolymers of glycolic acid derivatives, lactic acid derivatives, and p-dioxane derivatives. Etc. can be used. The polyalkylene glycol is preferably a polymer of oxyalkylene units having 2 to 4 carbon atoms, and those having an average polymerization number of 2 to 500 (preferably 45 to 500) can be used. The hydrophilic polymer is preferably a biocompatible polymer, and more preferably polyethylene glycol (PEG). The hydrophilic chain (b) may further have an arbitrary substituent.

As described above, the hydrophilic chain (b) has a functional group for linking to the surface of the base material by a covalent bond or the like (on the side opposite to the bonding position with the branched linker chain (c) in FIG. 1). It is preferable to have it at the terminal. As such a terminal functional group, for example, those shown below can be used (in the formula, the arrow represents the point of attachment to the hydrophilic chain (b)).

Preferably, an active ester group such as N-hydroxysuccinimide (NHS), a carboxyl group, a silanol group, a disulfide group, or a thiol group can be used as the terminal functional group. As will be described later, when a coating layer such as collagen is used on the substrate surface, functional groups capable of binding to these coating layers can be used. For example, in the case of a collagen coating layer, an amino group in collagen is used. An active ester group that can be covalently bonded to the olefin group is preferable, and an NHS group is particularly preferable.

As described above, the branched linker chain (c) is arranged between the hydrophobic chain (a) and the hydrophilic chain (b) and connects them, and in addition, photoisomerization as a side chain. It is a branched structure having a site (d). Linkage between the hydrophobic chain (a) and the hydrophilic chain (b) is, for example, shared amide bond, ester bond, ether bond, thioether bond, carbamate bond, thiocarbamate bond, triazole bond, urea bond, etc. Bonding can be used. The connection between the hydrophobic chain (a) and the branched linker chain (c) and the connection between the hydrophilic chain (b) and the branched linker chain (c) may be the same different bonding modes. It can be a different binding mode.

The material constituting the branched linker chain (c) is not particularly limited as long as it can link the hydrophobic chain (a) and the hydrophilic chain (b). It has a repeating structure. For example, an amino acid residue capable of introducing a branch such as lysine, aspartic acid, glutamic acid, cysteine, serine, threonine, tyrosine, or a repeating structure thereof is preferable. A lysine residue or a repeating structure thereof is particularly preferred. Alternatively, in addition to these amino acid residues, materials constituting the branched linker chain (c) include trihydric alcohols such as glycerol; benzenetriols such as hydroxyquinol and benzenetricarboxylic acids; benzenetriamines; 4-aminosalicylic acid, etc. A benzene ring having three or more reactive functional groups can also be used. In some cases, a hydrophilic polymer can be used in the same manner as the hydrophilic chain (b).

The photoisomerization site (d) is present in the side chain of the branched linker chain (c) and has a chemical structure that can change from hydrophobic to hydrophilic by changing the structure by isomerization by light irradiation. Is. Typically, in such photoisomerization, irradiation with light of a specific wavelength causes a reversible structural change caused by opening and closing of a cyclic structure, thereby changing the hydrophobicity of the chemical structure. Can be mentioned. Here, “reversible” causes a structural change such as ring opening (first isomerization reaction) by irradiation with light of a specific wavelength, and thereafter, light having a wavelength different from the specific wavelength. It is meant that a structural change (second isomerization reaction) occurs by re-irradiating or heating, and it is possible to return to the original closed ring structure. As a typical example, the photoisomerization site (d) is subjected to the first isomerization reaction by irradiation with ultraviolet light, and then the second isomerization reaction is performed by irradiation with visible light or heating. It has a chemical structure that occurs.

By photoisomerization of the photoisomerization site (d), a region consisting of a hydrophobic chain (a) and a branched linker chain (c) (including the photoisomerization site (d)) (that is, photoresponsive cell immobilization) It is desirable that the logarithmic value (log P) of the distribution coefficient in the region excluding the hydrophilic chain (b) in the agent is changed by a certain amount before and after the photoisomerization. Here, “log P” is a logarithmic value of the 1-octanol / water partition coefficient (P) of the compound, and the partition when the compound is dissolved as a solute in the two-liquid solvent system of 1-octanol and water. In equilibrium, it means the ratio of the equilibrium concentration of solutes in each solvent and is generally indicated in logarithmic form relative to the base 10. This log P is an index of hydrophobicity (lipophilicity). The larger the value, the more hydrophobic, and the smaller the value, the more hydrophilic. In the present invention, either an actual measured value or a calculated value of logP may be used. However, when there is an actual measured value, it is preferable to use the actual measured value. Note that the calculated value of logP is calculated by, for example, Hansch, Leo's fragment approach (A. Leo, Comprehensive Medicinal ｈ Chemistry, Vol. , Eds., P. 295, Pergamon Press, 1990), it is known that the program “CLOGP” (Daylight Chemical ｆ Information ｙ Systems, Inc.) can be calculated. Also, calculate the calculated value of logP using the calculation program ALOGPS 2.1 (Tetko, I. V. et al., J. Comput. Aided Mol. Des., 2005, 19 (6),） 453-463). You can also.

When the logP is changed by photoisomerization of the photoisomerization site (d) in the photoresponsive cell fixing agent of the present invention to form a hydrophilic isomer structure, the hydrophobic chain (a) On the other hand, when the target cell can be immobilized in the cell, on the other hand, when it becomes a hydrophobic isomer structure, it becomes possible to control the operation of the target cell detaching from the photoresponsive cell fixing agent. . This is because, when the photoisomerization site (d) has a hydrophobic structure, the regions of the hydrophobic chain (a) are densely packed together to inhibit the interaction with the target cell. When (d) has a hydrophilic structure, the degree of freedom of the region of the hydrophobic chain (a) is increased, and it is considered that the interaction with the target cell occurs and can be immobilized. From the viewpoint of such control, the amount of change in the logarithmic value (logP) of the distribution coefficient in the region consisting of the hydrophobic chain (a) and the branched linker chain (c) due to isomerization of the photoisomerization site (d) is 0. .5 or more is preferable. The amount of change is more preferably 0.8 or more, and particularly preferably 1.0 or more.

Furthermore, from the viewpoint of effectively immobilizing target cells in the hydrophobic chain (a), a region consisting of the hydrophobic chain (a) and the branched linker chain (c) when taking a hydrophobic isomer structure It is preferable that the logarithmic value (logP) of the partition coefficient in the surface has a hydrophobicity of 10 or more. That is, it is preferable that the logarithmic value (logP) of the partition coefficient of the isomer having higher hydrophobicity before and after the isomerization of the photoisomerization site (d) is 10 or more. More preferably, the logarithmic value (log P) of the partition coefficient of the more hydrophobic isomer is in the range of 10.5 to 11.0.

Therefore, in a preferred embodiment, the photoresponsive cell fixing agent of the present invention has a change in logarithmic value (logP) of the partition coefficient due to isomerization of the photoisomerization site (d) of 0.5 or more; and The logarithmic value (logP) of the partition coefficient in the case of a more hydrophobic isomer before and after the isomerization of the photoisomerization site (d) can be 10 or more.

Preferable examples that can be used as the photoisomerization site (d) that can provide such characteristics include, but are not limited to, those containing the structure of spiropyran or a derivative thereof. . As shown in the following formula, spiropyran has a relatively high hydrophobicity in the case of the closed ring structure on the left side of the formula, but the spiro ring portion is opened by a photoopening reaction upon irradiation with ultraviolet rays, and the open on the right side of the formula. It becomes a ring structure. The ring-opened structure has an intramolecular charge by electrolysis and has hydrophilicity. On the other hand, by irradiating visible light (or adding heat) to the ring-opened structure, the ring-closed structure can be restored again. That is, the hydrophobicity / hydrophilicity can be controlled by irradiation with light of a specific wavelength.

More specifically, the photoisomerization site (d) preferably has a spiropyran-like structure represented by the following formula (I).

In which R ₁ is a hydrogen atom or an alkyl group; R ₂ is a C ₁ -C ₂₀ alkylene group; R _A , R _B , and R _C may be the same or different and are each independently And a hydrogen atom, an alkyl group, an alkoxy group, or a nitro group; * is a binding moiety to the branched linker chain.

R ₁ is preferably a hydrogen atom or a C ₁ -C ₆ alkyl group, preferably a hydrogen atom. From the viewpoint of adjusting to appropriate hydrophobicity, R ₂ is preferably a C ₅ to C ₂₀ , more preferably a C ₅ or C ₆ linear alkylene group. Similarly, at least one of R _A , R _B , and R _C is preferably a nitro group, and R _C is more preferably a nitro group. By having the nitro group, the ring-opening structure by photoisomerization is stabilized, and the quantum yield of photoisomerization can be improved.

In the present specification, the “alkyl or alkyl group” may be any of an aliphatic hydrocarbon group composed of linear, branched, cyclic, or a combination thereof. The number of carbon atoms of the alkyl group is not particularly limited. For example, the number of carbon atoms is 1 to 20 (C _1-20 ), the number of carbons is 1 to 15 (C _{1 to 15} ), and the number of carbon atoms is 1 to 10 (C _{1 to 10).} ). In this specification, the alkyl group may have one or more arbitrary substituents. For example, C _1-8 alkyl includes methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neo-pentyl, n-hexyl, isohexyl, n-heptyl, n-octyl and the like are included. Examples of the substituent include an alkoxy group, a halogen atom (which may be a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom), an amino group, a mono- or di-substituted amino group, a substituted silyl group, or Although acyl etc. can be mentioned, it is not limited to these. When the alkyl group has two or more substituents, they may be the same or different. The same applies to the alkyl part of other substituents containing an alkyl part (for example, an alkoxy group, an arylalkyl group, etc.).

In the present specification, “alkylene” is a divalent group consisting of a linear or branched saturated hydrocarbon, such as methylene, 1-methylmethylene, 1,1-dimethylmethylene, ethylene, 1-methylethylene, 1-ethylethylene, 1,1-dimethylethylene, 1,2-dimethylethylene, 1,1-diethylethylene, 1,2-diethylethylene, 1-ethyl-2-methylethylene, trimethylene, 1 -Methyltrimethylene, 2-methyltrimethylene, 1,1-dimethyltrimethylene, 1,2-dimethyltrimethylene, 2,2-dimethyltrimethylene, 1-ethyltrimethylene, 2-ethyltrimethylene, 1,1 -Diethyltrimethylene, 1,2-diethyltrimethylene, 2,2-diethyltrimethylene, 2-ethyl-2-methyltrime Len, tetramethylene, 1-methyltetramethylene, 2-methyltetramethylene, 1,1-dimethyltetramethylene, 1,2-dimethyltetramethylene, 2,2-dimethyltetramethylene, 2,2-di-n-propyl Trimethylene and the like.

As used herein, “amide or amide group” refers to both RNR′CO— (in the case of R = alkyl, alkylaminocarbonyl-) and RCONR′— (in the case of R = alkyl, alkylcarbonylamino-). including.

In the present specification, when a functional group is defined as “may be substituted”, the type of substituent, the position of substitution, and the number of substituents are not particularly limited, and two or more When they have a substituent, they may be the same or different. Examples of the substituent include, but are not limited to, an alkyl group, an alkoxy group, a hydroxyl group, a carboxy group, a halogen atom, a sulfo group, an amino group, an alkoxycarbonyl group, and an oxo group. These substituents may further have a substituent. Examples of such include, but are not limited to, a halogenated alkyl group.

Specific examples of the photoresponsive cell fixing agent of the present invention include compounds having the following structures.

In the formula, Sp represents the photoisomerization site (d); m is a natural number of 1 to 5; and n is a natural number of 50 to 500. The compound comprises an oleyl group as the hydrophobic chain (a); a polyethylene glycol chain as the hydrophilic chain (b); a lysine residue as the branched linker chain (c); and a hydrophilic chain for binding to the substrate. (B) has N-hydroxysuccinimide at the terminal. Preferably, m is 1 or 2. In addition, Sp is preferably spiropyran or a derivative thereof, more preferably a structure represented by the above formula (I).

2. The present invention also relates to a cell immobilization substrate having a surface modified with the above-mentioned photoresponsive cell immobilization agent. As described above, the surface structure of the cell immobilization substrate is obtained by binding a photoresponsive cell immobilizing agent directly to the substrate surface at the end of the hydrophilic chain (b) or via a coating layer described later. is there. The photoresponsive cell fixing agent is preferably arranged in the form of a monolayer on the surface of the substrate.

The material and shape of the base material modified by the photoresponsive cell fixing agent are not particularly limited, and various appropriate base materials can be selected depending on the application. For example, the shape of the base material to be modified may be a substrate (plate or film, such as a glass slide, dish, microplate, microarray substrate, etc.), Colloidal), fibrous structures, tubes, containers (eg test tubes and vials). Materials for the base material to be modified include glass; cement; ceramics or fine ceramics such as ceramics; polymer resins such as polyethylene terephthalate, cellulose acetate, polycarbonate, polystyrene, and polymethyl methacrylate; biomaterials such as polypeptides and proteins; silicon; Examples include activated carbon; porous glass; porous ceramics; porous silicon; porous activated carbon; non-woven fabric; filter paper; membrane filter; Since the surface of the base material to be modified introduces amino groups, carboxyl groups, hydroxy groups, etc., it can be coated with a polymer such as polycation or treated with a silane coupling agent having a substituent introduced onto the surface of the base material. It may be applied or a reactive functional group may be introduced by plasma treatment.

The photoresponsive cell fixing agent may be modified by directly bonding to the substrate surface, or a coating layer is provided on the substrate surface, and the photoresponsive cell fixing agent is provided on the surface of the coating layer. Surface modification can also be performed by bonding. As such a coating layer, for example, collagen, bovine serum albumin (BSA), 3-aminopropyltriethoxysilane (APTES), or ovalbumin can be used.

3. Cell immobilization and recovery method Further, the present invention provides a cell sorting technique for immobilizing and selectively recovering target cells using a cell immobilization substrate surface-modified with a photoresponsive cell immobilization agent. Related. The cell recovery method of the present invention includes the following steps:
(I) irradiating the cell-immobilizing substrate surface-modified with the photoresponsive cell fixing agent of the present invention with light of a specific wavelength, and converting the photoisomerization site into a hydrophilic isomer structure;
(Ii) a step of bringing a solution containing predetermined target cells into contact with the substrate for cell immobilization, and immobilizing the target cells on the substrate for cell immobilization; and (iii) the substrate for cell immobilization. Is irradiated with light having a wavelength different from that in the step (i), and the photoisomerization site is converted into a hydrophobic isomer structure, whereby the immobilized target cells are removed from the cell immobilization substrate. Separation / recovery process.

In the step (i), irradiation with light of a specific wavelength such as ultraviolet light is performed to isomerize the photoisomerization site, thereby increasing hydrophilicity and the target cell by the hydrophobic chain in the photoresponsive cell fixing agent. It is in a state where it can be fixed. For example, a ring-opened state having an intramolecular charge in the spiropyran structure corresponds to this.

On the other hand, in step (iii), irradiation of light such as visible light causes the photoisomerization site to have a hydrophobic structure, thereby eliminating the immobilization of the target cell by the hydrophobic chain. Can be selectively recovered at the single cell level. For example, the closed ring state in the spiropyran structure corresponds to this.

Since the photoresponsive cell fixing agent having the hydrophobic structure in the step (iii) can be re-irradiated with the light by re-irradiating the photoresponsive cell fixing agent, the following repetitive operation is performed. Can do. If the photoisomerization site has a spiropyran structure, the light irradiated in the step (i) is ultraviolet light; the light irradiated in the step (iii) is visible light. Is preferred.

Typically, as a place for performing the method, a cell immobilization substrate can be installed in the microchannel. In this case, in the step (iii), a flux can be imparted to the surface of the substrate, and the target cells separated from the cell immobilization substrate can be recovered.

By repeating the above steps (i) to (iii) a plurality of times, the target cells are continuously immobilized and recovered only by light irradiation without special treatment of the cell immobilization substrate itself. It becomes possible.

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

1. Reagents and equipment, etc.
[Equipment etc.]
Unless otherwise stated, the chemicals used in this example were analytical grade and were used without further purification. 2,3,3-trimethylindolenine, 6-bromohexanoic acid, 5-bromovaleric acid, oleic acid, 3-iodopropionic acid, sodium nitrite, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride Salt (EDC), 3-methyl-2-butanone (MIPK) and N, N′-dicyclohexylcarbodiimide (DCC) were obtained from Tokyo Chemical Industry. p-Butylaniline, 7-bromoheptanoic acid, 2-hydroxy-5-nitrobenzaldehyde, Nα-Boc-L-lysine, N-hydroxysuccinimide (NHS), triethylamine (TEA), tin (II) chloride N, N ' -Diisopropylcarbodiimide (DIC), sodium iodide and hydrochloric acid were obtained from Wako Pure Chemical Industries. Normal PEG lipid (Oleyl-PEG-NHS, Sunbright OE-040CS) and bifunctional polyethylene glycol (H ₂ N-PEG-COOH, Sunbright PA-034HC) were obtained from NOF. Trifluoroacetic acid (TFA) was obtained from Watanabe Chemical Industry. Dehydrated TEA was prepared by distillation.

[apparatus]
Column chromatography was performed using silica gel (Kanto Chemical) (60N sphere, 40-50 μm). TLC was performed on glass support plates coated with silica gel (60, F254) (Merck). NMR chemical shifts are given in ppm using tetramethylsilane (TMS) as an internal reference. For the NMR spectrum, Avance 600 (600 MHz, Bruker) was used. For ESI mass spectrum, micrOTOF II (Bruker) was used. For MALDI-TOF spectra, Microflex (matrix: disranol and NaCl) was used.

[Cell culture, etc.]
Ba / F3 murine IL-3-dependent pro-B cell line, K562 human erythroleukemia, Jurkat human T-cell leukemia and HeLa human epithelial cancer were obtained from RIKEN BioResource Center. Enhanced green fluorescent protein (EGFP) -expressing Ba / F3 cells were prepared according to the literature. RPMI-1640 medium was obtained from Wako Pure Chemical. Fetal bovine serum is from Thermo Fisher Scientific. Mouse interleukin (IL) -3,0.25% trypsin / EDTA solution and Dulbecco's modified Eagle medium were obtained from Gibco BRL. Penicillin / streptomycin and trypan blue staining solutions were obtained from Nacalai Tesque. Collagen was obtained from Nitta Gelatin. CalceinAM was obtained from Dojin Research Laboratory. PBS- was purchased from Nissui Pharmaceutical.

2. Synthesis of Photoresponsive Cell Fixing Agent Compound 1 which is a photoresponsive cell fixing agent of the present invention was synthesized according to the following scheme.

2-1. Synthesis of Compound 2 Oleic acid (340 μl), EDC (612.5 mg) and NHS (167.7 mg) were dissolved in anhydrous dichloromethane (DCM) and reacted at o / n. Purification on a silica column (DCM) gave compound 2 as a clear oil (354.3 mg, 88%).
TLC (DCM) Rf: 0.55 (in iodine)
ESI-MS (m / z): [M + Na ⁺ ] calculated 334.08; observed 334.06.
¹ H-NMR (600 MHz, DMSO-d6, TMS): δ 0.85 (t, 3H), 1.24 (m, 20H), 1.60 (quint, 2H), 1.98 (q, 4H), 2.64 (t, 2H) , 2.80 (s, 4H), 5.32 (t, 2H).

2-2. Synthesis of Compound 3 Compound 2 (98.6 mg), Nα-Boc-L-lysine (83.5 mg) and anhydrous TEA (50 μl) were dissolved in anhydrous DCM and reacted at o / n. The reaction mixture was first extracted with EtOAc and water (pH 2), then silica column (DCM / MeOH = 10/1) gave compound 3 as a yellow solid (126.3 mg, 95%).
TLC (DCM) Rf: 0.65 (in iodine)
ESI-MS (m / z): [M + Na ⁺ ] calculated 533.39; observed 533.43.
¹ H-NMR (600 MHz, DMSO-d6, TMS): δ 0.85 (t, 3H), 1.23 (m, 24H), 1.37 (s, 9H), 1.45 (t, 2H), 1.53 (br, 2H) , 1.97 (q, 4H), 2.01 (t, 2H), 2.99 (q, 2H), 3.80 (m, 1H), 5.32 (q, 2H), 7.02 (d, 1H), 7.73 (t, 1H), 12.41 (br, 1H).

2-3. Synthesis of Compound 4 Compound 3 (126 mg), EDC (292.1 mg) and NHS (73.8 mg) were dissolved in anhydrous DCM and reacted at o / n. Silica column (DCM / acetone = 5/1) gave compound 4 as a white solid (122.6 mg, 82%).
TLC (DCM / MeOH = 5/1) Rf: 0.6 (in iodine)
ESI-MS (m / z): [M + Na +] calculated 630.40; observed 630.36.
¹ H-NMR (600 MHz, DMSO-d6, TMS): δ 0.85 (t, 3H), 1.23 (br, 22H), 1.38 (s, 11H), 1.46 (quint, 2H), 1.98 (br, 2H) , 2.02 (m, 6H), 2.80 (br, 4H), 3.00 (q, 2H), 4.29 (br, 1H), 5.32 (quint, 2H), 7.58 (d, 1H), 7.74 (t, 1H).

2-4. Synthesis of Compound 5 Compound 4 (578.7 mg), PA-034HC (1.5 g) and anhydrous TEA (200 μl) were dissolved in anhydrous DCM and reacted for 2 days. The reaction mixture was concentrated and added dropwise to ice-cold ether. The resulting precipitate was collected using centrifugation (15 kG, 10 min, −10 ° C.) and then dissolved in water. The by-product was removed by dialysis (MWCO = 2 kDa) and lyophilized to give compound 5 as a white solid (1.6226 g, 95%).
TLC (DCM / MeOH = 8/1) Rf: 0.25 (in iodine)
¹ H-NMR (600 MHz, DMSO-d6, TMS): δ 0.85 (t, 3H), 1.14 (br, 24H), 1.37 (s, 9H), 1.46 (m, 6H), 1.60 (quint, 2H) , 1.98 (m, 6H), 2.19 (t, 2H), 2.80 (br, 2H), 2.98 (m, 2H), 3.50 (s, approx. 300H), 3.62 (t, 2H), 3.79 (br, 1H ), 5.32 (t, 2H), 6.72 (d, 1H), 7.72 (t, 1H), 7.76 (t, 1H), 11.99 (br, 1H).

2-5. Synthesis of Compound 8 2,3,3-Trimethylindolenine (Compound 7) (3 ml, 18.727 mmol) and 3-iodopropanoic acid (4.5086 g, 22.546 mmol) were dissolved in 30 ml of anhydrous 2-butanone, The mixture was heated to reflux for 4 days under an Ar atmosphere. After cooling, the resulting precipitate was collected by filtration and washed with EtOAc to give compound 8 as a light brown powder (3.5439 g, 82%).
TLC (DCM / MeOH = 10/1) Rf: 0.05 (in UV)
ESI-MS (m / z): [M ⁺ ] found 232.13; observed 232.25.
¹ H-NMR (600 MHz, DMSO-d6, TMS): δ 1.52 (s, 6H), 2.85 (s, 3H), 2.97 (t, 2H), 4.64 (t, 2H), 7.61 (d, 2H) , 7.83 (d, 1H), 7.99 (d, 1H).

2-6. Synthesis of Compound 9 Compound 8 (511.2 mg), 2-hydroxy-5-nitro-benzaldehyde (372.2 mg) and TEA (300 μl) were dissolved in 5 ml of anhydrous 2-butanone and heated to reflux for 3 hours. After cooling, the reaction mixture was first extracted with DCM / water and then compounded as a reddish brown solid (590.5 mg, 74%) by silica column purification (DCM / MeOH = 15/1 to 1/1/1% triethylamine) 9 was obtained.
TLC (DCM / MeOH = 10/1) Rf: 0.45 (in UV)
ESI-MS (m / z): [M + H ⁺ ] calculated 381.14; observed 381.28.
¹ H-NMR (600 MHz, DMSO-d6, TMS): δ 1.07 (s, 3H), 1.18 (s, 3H), 2.46 (m, 1H), 2.55 (m, 1H), 3.35 (m, 1H) , 3.48 (m, 1H), 6.00 (d, 1H), 6.67 (d, 1H), 6.80 (t, 1H), 6.87 (d, 1H), 7.12 (m, 2H), 7.20 (d, 1H), 7.99 (dd, 1H), 8.22 (d, 1H), 12.24 (br, 1H).

2-7. Synthesis of Compound 10 Compound 9 (220.7 mg, 0.512 mmol), EDC (311.5 mg, 1.625 mmol) and NHS (119.9 mg, 1.041 mmol) are dissolved in 5 ml of anhydrous DCM and stirred for 2 hours under Ar atmosphere. did. The reaction mixture was extracted with DCM / water (pH 7) and dried over MgSO4 to give compound 10 as a brown solid (245.0 mg, 99%).
TLC (EtOAc) Rf: 0.85 (in UV)
ESI-MS (m / z): [M + H ⁺ ] calculated 478.16; observed 478.30.
¹ H-NMR (600 MHz, DMSO-d6, TMS): δ 1.16 (s, 3H), 1.24 (s, 3H), 2.86 (br, 4H), 3.00 (m, 1H), 3.14 (m, 1H) , 3.50 (m, 1H), 3.64 (m, 1H), 6.10 (d, 1H), 6.83 (d, 1H), 6.91 (t, 1H), 6.96 (d, 1H), 7.21 (m, 2H), 7.27 (d, 1H), 8.05 (dd, 1H), 8.28 (d, 1H).

2-8. Synthesis of Compound 6 Compound 5 (58.7 mg, 0.01508 mmol) was dissolved in a mixture of 2 ml DCM and 1 ml TFA and stirred for 15 minutes. Concentration and vacuum drying gave compound 6 as a white oil (obtained as a mixture with TFA). The reaction mixture was used in the next reaction without further purification.
TLC (DCM / MeOH = 8/1) Rf: 0.15 (in ninhydrin)
¹ H-NMR (600 MHz, DMSO-d6, TMS): δ 0.85 (t, 3H), 1.24 (br, 24H), 1.38 (q, 2H), 1.47 (m, 6H), 1.69 (br, 4H) , 1.98 (q, 6H), 2.19 (t, 2H), 3.00 (br, 2H), 3.50 (s, approx. 300H), 5.32 (t, 2H), 7.87 (br, 1H), 8.28 (br, 3H ), 8.62 (br, 1H), 10.34 (br, 1H).

2-9. Synthesis of Compound 11 Compound 10 (48.0 mg, 0.101 mmol), Compound 6 (98.7 mg, 0.0115 mmol) and anhydrous TEA (200 μl, 1.4366 mmol) were dissolved in 5 ml of anhydrous DCM and allowed to stand for 3 days under an Ar atmosphere. Stir. The reaction mixture was concentrated and added dropwise to ice-cold ether. The resulting precipitate was collected using centrifugation (15 kG, 10 min, −10 ° C.) and then dissolved in water. The by-product was removed by dialysis (MWCO = 2 kDa) and lyophilized to give compound 11 as a brown solid (43.5 mg, 92%).
TLC (DCM / MeOH = 5/1) Rf: 0.7 (in UV)
¹ H-NMR (600 MHz, DMSO-d6, TMS): δ 0.85 (t, 3H), 1.23 (br, 26H), 1.28 (t, 4H), 1.47 (m, 8H), 1.60 (m, 2H) , 1.97 (m, 6H), 2.18 (t, 2H), 3.06 (br, 4H), 3.50 (s, approx. 300H), 3.62 (s, 2H), 4.02 (m, 2H), 5.31 (t, 2H ), 6.00 (br, 1H), 6.66 to 8.21 (br, 10H), 11.98 (br, 1H).

2-10. Synthesis of Compound 1 Compound 11 (12.7 mg, 0.0031 mmol), DIC (50 μl, 0.3229 mmol) and NHS (6.5 mg, 0.0564 mmol) are dissolved in 5 ml of anhydrous DCM and stirred for 2 days under Ar atmosphere. did. The reaction mixture was concentrated and added dropwise to ice-cold ether. The resulting precipitate was collected using centrifugation (15 kG, 10 minutes, −10 ° C.) and dried in vacuo to give compound 1 as an orange solid (12.1 mg, 93%).
TLC (DCM / MeOH = 8/1) Rf: 0.3 (in UV)
¹ H-NMR (600 MHz, DMSO-d6, TMS): δ 0.85 (t, 3H), 1.23 (br, 26H), 1.37 (t, 4H), 1.45 (m, 4H), 1.51 (m, 2H) , 1.61 (m, 4H), 1.97 (m, 6H), 2.66 (t, 2H), 2.81 (br, 4H), 2.92 (br, 4H), 3.50 (s, approx. 300H), 4.06 (m, 2H ), 5.31 (t, 2H), 6.01 to 8.52 (m, 11H).

Using a reaction similar to that of Compound 1, Compound Groups 1a to 1e shown in Table 1 below, in which the substituents of R ₁ and R _{2 at} the spiropyran moiety are different, were respectively synthesized.

3. 2. Verification of change in log P due to photoisomerization The logarithmic value of the partition coefficient (logP) in the region consisting of a hydrophobic chain and a branched linker chain accompanying the structural change of the spiropyran moiety due to photoisomerization of the compound group that is the photoresponsive cell fixing agent of the present invention synthesized in (1). ) Was calculated. The calculation is performed by Igor V. Tetko et al., ALOGPS 2.1 (Tetko, IV; Gasteiger, J .; Todeschini, R .; Mauri, A .; Livingstone, D .; Ertl, P .; Palyulin, VA; Radchenko, EV Zefirov, NS; Makarenko, AS; et al. J. Comput. Aided. Mol. Des. 2005, 19 (6), 453-463). The results are shown in Table 2 below.

As a result, it was found that the log P value is greatly reduced by isomerization from the closed state (Closed form) to the open state (Open form), that is, the compound changes from hydrophobic to hydrophilic. . Regarding the amount of change, the difference in the log P value between the closed state (Closed form) and the open state (Open form) is 1.0 or more, and it was found that the hydrophobicity can be greatly changed by photoisomerization. In addition, the log P value in the closed state (Closed form) is 10.00 or more, and has an appropriate hydrophobicity that does not inhibit the hydrophobic interaction with cells in the oleyl chain region (hydrophobic chain). I understood.

4. Substrate preparation The surface of a glass substrate was modified with a photoresponsive cell fixing agent by the following procedure.

4-1. Modification of substrate with photoresponsive cell fixing agent A glass bottom dish having a diameter of 35 mm was used. A collagen solution (3 mg / ml) was diluted 10-fold with MQ (pH 3) to adjust to 0.3 mg / ml, 1 ml was added to each dish, and allowed to stand at room temperature overnight. After washing 3 times with 2 ml of MQ, excess MQ was removed with a pipetman and air-dried until water droplets disappeared completely from the surface. Dilute the stock of photoresponsive cell fixing agent (10 mM in DMSO) to the desired concentration with PBS, one sample of positive control, two samples of photoresponsive cell fixing agent on the diagonal, PBS- as negative control A total of four spots were spotted on each dish at 0.3 μl. After incubating at 37 ° C. for 1 hour, the cells were washed once with 1 ml of TrisHCl (50 mM, pH 7.5), then 5 times with 2 ml of MQ, and allowed to stand in 1 ml of PBS-.

4-2. Immobilization of cells on the modified substrate The cells prepared in (1) were immobilized on a substrate by the following procedure. After irradiating the dish surface with PBS (360 nm or 520 nm, 10 min) in PBS-, remove PBS-, add 200 μl of cell suspension and gently shake. The mixture was allowed to stand at room temperature for 10 min. After washing 3 times with 1.5 ml PBS-, 1.5 ml PBS- was added.

5. Cell preparation 5-1. Thaw, culture, and freeze cells
The thawed serum-containing medium was warmed in a 37 ° C thermostatic bath, and the frozen cells were thawed in the thermostatic bath until some ice remained. As soon as it was completely dissolved, it was transferred to a 15 ml tube and diluted with 9 ml of medium. The supernatant was removed by centrifugation (190 G, 3 min, hereinafter the same), and the suspension was resuspended in 10 ml of medium and spread on a 100 mm dish.
Cultivation Basically culturing in an incubator (37 ° C, 5% CO ₂ ). The medium composition is as follows.
eGFP expression Ba / F3: RPMI (10% FBS), 1 ng / ml IL3
Passage Ba / F3 is only diluted with basic fresh medium.
Frozen cells are collected in a 15 ml tube, washed once with clean medium, suspended in Cell Banker, dispensed in 0.5 or 1 ml aliquots, and stored at -80 ° C. Long-term storage samples were stored in liquid nitrogen.

5-2. Pretreatment of cells for cell fixation Ba / F3 was collected by centrifugation (100 G, 3 min, hereinafter the same), then washed twice with 10 ml PBS- to 3 × 10 ⁶ cells / ml. It was resuspended in PBS-.

6). Cell fixing force (light switching) evaluation Next, the cell fixing ability accompanying light irradiation was evaluated using a substrate modified with a photoresponsive cell fixing agent.

6-1. Evaluation of Cell Fixation Strength of PEG Lipid 2. Cell suspension (mouse proB cell BaF3 strain) acts on the substrate modified with the photoresponsive cell immobilization agent according to the present invention synthesized in step 3 after irradiation with light in a wavelength range that promotes the opening and closing of spiropyran. As a result, changes in cell fixing force were observed. The results obtained for compounds 1, 1b, 1c, 1d, and 1e are shown in FIGS. 2 (a) to 2 (e), respectively. The vertical axis represents the number of cells immobilized on the substrate, and the horizontal axis represents the concentration of the photoresponsive cell fixing agent. At the same concentration, the left side shows the result after ultraviolet irradiation (Open form), and the right side shows the result after visible light irradiation (closed form). The number of cells was manually counted from an image taken with a confocal microscope.

As a result, the cells were fixed relatively strongly on the surface of the spiropyran derivative ring-opened by irradiation with ultraviolet light (360 nm), and conversely on the surface closed by irradiation with visible light (520 nm). It was observed that the cells were relatively weakly fixed. In particular, it was found that in the substrate using the compound 1d having the most excellent switching ability, the cells are fixed by irradiation with ultraviolet light, but the cells are not substantially fixed by irradiation with visible light. By optimizing the chemical structure of such a light-switching cell fixing agent, it was demonstrated that a surface capable of switching the cell fixing force can be obtained by irradiating light of different wavelengths.

6-2. Verification of repeated measurement By making the ring-opened state of compound 1d-modified surface in a closed ring state by irradiation with ultraviolet light, cells are once fixed on the substrate surface and continuously irradiated with visible light. It was confirmed that the fixed cells can be detached by (FIG. 3).

In addition, it was confirmed that cell fixation and detachment could be repeated reversibly by repeatedly irradiating ultraviolet light and visible light alternately (FIG. 4). Furthermore, as shown in FIG. 5, it was confirmed that this switching step can be performed with high spatial resolution by performing light irradiation on a specific region on the substrate.

7. Immobilization with other cells The Ba / F3 cells used in Example 1 were floating cells, but the same experiment was performed to show that other floating cells could be similarly reversibly immobilized (compound 1d was used). The cells used were K562 cells and Jurkat cells, both of which are human leukemia-derived cell lines. Experiments were also performed on suspended adherent cells (HeLa cells (human cervical cancer-derived cell line)). The obtained results are shown in FIG. 6 ((a) K562 cells (b) Jurkat cells (c) HeLa cells). In the figure, “off” is the cell immobilization density after irradiation with visible light, and “on” is the cell immobilization density after ultraviolet irradiation. From the result of FIG. 6, it was demonstrated that reversible cell immobilization can be achieved for various cells by using the photoresponsive cell fixing agent of the present invention.

8. Cytotoxicity verification The cell viability (Released) once fixed on the substrate and then removed by irradiation with visible light, and the viability of control cells in PBS for the same time (Pos. Ctrl.) The results of comparison are shown in FIG. 7 (compound 1d was used). As a result, it was confirmed that the operation of “fixing once on the substrate and then removing it from the substrate” has no toxicity to the cells.

Claims

A photoresponsive cell fixing agent for fixing a predetermined target cell on a substrate,
A hydrophobic chain capable of interacting with the target cell;
A hydrophilic chain that can be arranged in the form of a monomolecular film on the surface of the substrate, and a branched linker chain that connects the hydrophobic chain and the hydrophilic chain;
The photoresponsive cell immobilizing agent characterized in that a side chain of the branched linker chain has a photoisomerization site which can be isomerized by light irradiation and change from hydrophobic to hydrophilic.
The amount of change in the logarithmic value (logP) of the distribution coefficient due to the isomerization of the photoisomerization site is 0.5 or more (where the distribution coefficient includes the hydrophobic chain (a) and the branched linker chain (c ), And the photoresponsive cell fixing agent according to claim 1.
The photoresponsive cell fixing agent according to claim 2, wherein the logarithmic value (log P) of the partition coefficient in the more hydrophobic isomer before and after isomerization of the photoisomerization site is 10 or more.
The photoresponsive cell fixing agent according to claim 1, wherein the photoisomerization site contains a structure of spiropyran or a derivative thereof.
The photoresponsive cell fixing agent according to claim 1, wherein the photoisomerization site has a structure represented by the following formula (I).

(Wherein R 1 is a hydrogen atom or an alkyl group; R 2 is a C 1 to C 20 alkylene group; R A , R B , and R C may be the same or different; Independently, a hydrogen atom, an alkyl group, an alkoxy group, or a nitro group; * is a binding moiety to the branched linker chain.)
The photoresponsive cell fixing agent according to claim 5, wherein R 2 is a C 5 or C 6 linear alkylene group; and R C is a nitro group.
The photoresponsive cell fixing agent according to any one of claims 1 to 6, wherein the hydrophobic chain is a saturated or unsaturated hydrocarbon chain which may have a substituent.
The photoresponsive cell fixing agent according to any one of claims 1 to 7, wherein the hydrophilic chain comprises a hydrophilic polymer.
The photoresponsive cell fixing agent according to claim 8, wherein the hydrophilic polymer is polyalkylene glycol.
The photoresponsive cell fixing agent according to any one of claims 1 to 9, wherein the branched linker chain has an amino acid residue or a repeating structure thereof.
The photoresponsive cell fixing agent according to any one of claims 1 to 10, wherein the photoisomerization site is linked to the branched linker chain through an amide bond.
The photoresponsive cell fixing agent according to any one of claims 1 to 11, which has a substituent capable of being covalently bonded to the surface of the substrate at the end of the hydrophilic chain.
The photoresponsive cell fixing agent according to claim 1, which has the following structure.

(In the formula, Sp represents the photoisomerization site; m is a natural number of 1 to 5; and n is a natural number of 50 to 500.)
A cell immobilization substrate having a surface modified with the photoresponsive cell immobilization agent according to any one of claims 1 to 13.
(I) irradiating the cell-immobilizing substrate according to claim 14 with light of a specific wavelength, and converting the photoisomerization site into a hydrophilic isomer structure;
(Ii) a step of bringing a solution containing predetermined target cells into contact with the substrate for cell immobilization, and immobilizing the target cells on the substrate for cell immobilization; and (iii) the substrate for cell immobilization. Is irradiated with light having a wavelength different from that in the step (i), and the photoisomerization site is converted into a hydrophobic isomer structure, whereby the immobilized target cells are removed from the cell immobilization substrate. Separation / recovery process,
A cell recovery method comprising:
The method for recovering cells according to claim 15, comprising repeating the steps (i) to (iii) a plurality of times.
The cell collection method according to claim 15 or 16, wherein the light irradiated in the step (i) is ultraviolet light; and the light irradiated in the step (iii) is visible light.