WO2021117680A1 - Biological substance immobilization material - Google Patents

Abstract

[Problem] The problem addressed is to develop a system that enables a biological substance such as cells to be rapidly and easily immobilized on a hydrogel thin film. [Solution] An immobilization material, for immobilizing a biological substance having a lipid membrane on a substrate, wherein: a hydrogel thin film layer, comprising a polymer, and a structure in which a biological substance immobilization compound is modified on the hydrogel thin film layer are provided; the biological substance immobilization compound comprises a hydrophobic chain that can interact with the biological substance and a hydrophilic chain that connects to the surface of the hydrogel thin film layer; a reactive group X that can bind to the surface of the hydrogel thin film by a covalent bond is provided at a terminal of the hydrophilic chain; and the polymer comprises, within the molecule, a reactive group Y that forms a covalent bond with the reactive group X.

Description

Biomaterial immobilization material

The present invention relates to a material for rapidly and easily immobilizing a biological substance having a lipid membrane, a cell immobilization base material having a surface modified by the material, and a cell recovery method using the base material.

It is known that in in vivo tissues, many cells adhere to and extend on the extracellular matrix to express normal functions, and form a three-dimensional tissue while being supported by the extracellular matrix from the surroundings. ing. Such extracellular matrix is a hydrogel containing fibrous proteins such as collagen and proteoglycan and polysaccharides containing water, and mainly functions as a scaffold for cells. For example, when culturing hepatocytes in vitro, the function is lost if the cells are directly adhered to the culture substrate and cultured, whereas the function can be maintained for a long period of time on the substrate coated with extracellular matrix gel. Many examples have been reported (for example, Non-Patent Document 1). In addition, the function of cells is controlled by the hardness of extracellular matrix, the contained humoral components, and the properties of the presented molecules, and the functionalization of cells such as matrix gel that promotes the formation of tubular structures of vascular endothelial cells Therefore, it has been reported that a specific extracellular matrix gel is essential (for example, Non-Patent Document 2). For this reason, in drug discovery and toxicity tests using the in vivo function of cells as an index, and in regenerative medicine using the in vivo function, cells are seeded on extracellular matrix gel and cultured, assayed, and organized. It has been.

On the other hand, in a wide range of research fields such as drug discovery, cell engineering, and tissue engineering, artificial synthetic polymer gels are used as functional cell culture carriers. In particular, hydrogels containing water as a solvent have a sustained release function of the contained molecules, so that many types of drugs arrayed in the gel are released at the same time, and the effect on the cells cultured on the gel. Is applied to a technique for comprehensively analyzing (Non-Patent Document 3). In addition, a technique for sensing cell secretions by modifying the polymer fibers in the gel with fluorescent sensor molecules (Non-Patent Document 4) and cells adhered to a photosoluble synthetic hydrogel are irradiated with light. Techniques for selective recovery (Non-Patent Document 5) and the like have been reported. As described above, the technique of culturing and assaying cells on a synthetic polymer gel is also widely applied.

Against this background, there is a need to develop a technique for seeding cells on the surface of hydrogel at high density, quickly and easily. However, as a conventional method for seeding cells on a hydrogel, a method of placing a cell suspension on the surface of the hydrogel and spontaneously adhering the cells to the gel material is generally used. However, such a method can be used only for adhesive cells, and has a problem that it cannot be applied to some weakly adhesive hepatocytes and many non-adhesive blood cells and immune cells. It was.

A method using an antibody has also been reported as a method for capturing floating cells on the gel surface (Non-Patent Document 6). However, antibodies are expensive to manufacture, and those with binding specificity are extremely expensive. In addition, it is difficult to obtain antibodies that can strongly recognize the cell membrane surface of any cell.

Further, as a method of arranging cells on hydrogel, a method of ejecting cells using an inkjet printer has also been reported (for example, Non-Patent Document 7). However, when ejected by an inkjet printer, cytotoxicity due to pressure or heat becomes a problem. In addition, it is currently difficult to accurately eject cells one by one, and even if they can be ejected one by one, it takes an enormous amount of time, so it is difficult to arrange cells with the precision of one cell level. .. Further, a technique of incorporating magnetic beads into cells and arranging the cells on a hydrogel using a fine magnet has also been reported (Non-Patent Document 8). However, microfabrication of magnets is not inexpensive, and there is a limit to the shape of the pattern that can be arranged. In addition, the magnetic beads taken up by the cells cannot be taken out after being placed, and there is concern about the influence on the cell assay and regenerative medicine.

On the other hand, the inventors of the present invention have already reported a method of transferring cells arranged on a substrate by pressing them onto a hydrogel (Non-Patent Document 9). However, when such a method is used, there is a problem that it can be applied only to cells having adhesiveness to hydrogel because adhesion between cells and the surface of hydrogel is required for transcription.

Toshiaki Takezawa et al., Pharmacy Magazine, 2008, 128, 51 Akhtar N, et al., Angiogenesis, 2002, 5, 75-80 ailey S. N., et al., Proc. Natl. Acad. Soc. USA, 2004, 101, 16144-816149 Son K. J., et al., Anal. Chem., 2013, 85, 11893-11901 Tamura M., et al., Sci. Rep., 2015, 5, 15060 Son K. J., et al., Lab Chip, 2015, 15, 637-641 Nahmias Y., et al., Tissue Eng., 2005, 11, 701-708 Okochi M., et al., Lab Chip, 2009, 9, 3378-3384 Takano T., et al., Biotechnol. Bioeng., 2012, 109, 244-251

Therefore, an object of the present invention is to solve the problems in the above-mentioned conventional method and to develop a system capable of quickly and easily immobilizing a biological substance such as a cell on a hydrogel thin film.

As a result of diligent studies to solve the above problems, the present inventors used a thin film of a hydrogel having a reactive group on the surface, and used a biomaterial-immobilized compound capable of interacting with a biomaterial to react on the surface of the hydrogel. It has been found that by modifying the sex group, any biological substance such as a cell can be rapidly fixed to the surface of the hydrogel. It was also found that cells and the like can be selectively immobilized or recovered by light irradiation by using a biological substance-immobilized compound or hydrogel having a photoreaction. These findings have led to the completion of the present invention.

That is, the present invention, in one aspect,
<1> An immobilization material for immobilizing a biological substance having a lipid film on a substrate, which has a hydrogel thin film layer made of a polymer and a structure in which a biological substance immobilizing compound is modified on the hydrogel thin film layer. The biomaterial-immobilized compound has a hydrophobic chain capable of interacting with the biomaterial and a hydrophilic chain linked to the surface of the hydrogel thin film layer; at the end of the hydrophilic chain, said. The immobilization material having a reactive group X that can be covalently bonded to the surface of the hydrogel thin film layer; the polymer having a reactive group Y in the molecule that forms a covalent bond with the reactive group X;
<2> The immobilization material according to <1> above, wherein the hydrophilic chain contains a polyalkylene glycol;
<3> The immobilization material according to <1> or <2> above, wherein the hydrophobic chain is a saturated or unsaturated hydrocarbon chain which may have a substituent;
<4> The combination of the reactive group X and the reactive group Y is an amino group and an N-hydroxy-succinimidyl (NHS) group; an amino group and a sulfosuccinimidyl group; an amino group and a nitrophenyl ester group; an azido group. Alkin group; azide group and dibenzocyclooctine group; thiol group and maleimide group; thiol group and iodoacetamide group; thiol group and vinylsulfone group; aldehyde group and hydrazine group; ketone group and hydrazine group; aldehyde group and aminooxy group; The immobilization material according to any one of <1> to <3>, which is selected from the group consisting of a ketone group and an aminooxy group;
<5> The immobilization material according to any one of <1> to <3> above, wherein the reactive group X is a nucleophilic functional group and the reactive group Y is an electrophilic functional group;
<6> The immobilization material according to any one of <1> to <3> above, wherein the reactive group X is an electrophilic functional group and the reactive group Y is a nucleophilic functional group;
<7> Any of the above <1> to <6>, wherein the sex polymer is a carbohydrate-based polymer, a protein-based polymer, a polyester hydroxy acid, a polyanhydride, a polyvinylpolyhydroxyalkylmethacrylate, a polyvinylpyrrolidone, a polyvinyl alcohol, or a polyamide. The immobilization material according to 1.
<8> The immobilization material according to any one of <1> to <7> above, wherein the polymer is polyethylene glycol (PEG) having two branches, four branches, or eight branches;
<9> The polyethylene glycol has a first polymer unit having one or more nucleophilic functional groups in the side chain or the terminal, and a second polymer unit having one or more nucleophilic functional groups in the side chain or the terminal. The immobilization material according to <8> above, wherein the polymer units are crosslinked with each other to form a hydrogel;
<10> The nucleophilic functional group is selected from the group consisting of a thiol group and an amino group; the electrophilic functional group is a maleimidyl group, an N-hydroxy-succinimidyl (NHS) group, a sulfosuccinimidyl. The immobilization material according to any one of <5> to <9>, which is selected from the group consisting of a group, a phthalimidyl group, an imidazolyl group, an acryloyl group, -CO ₂ PhNO _{2, and a nitrophenyl group;}
<11> The polymer is polyethylene glycol (Tetra-PEG) having four branches; the nucleophilic functional group is an amino group; the electrophilic functional group is N-hydroxy-succinimidyl (NHS). The immobilization material according to any one of <5> to <10>, which is a group;
<12> The immobilization material according to <11> above, wherein the reaction group X is an N-hydroxy-succinimidyl (NHS) group and the reaction group Y is an amino group;
<13> The immobilization material according to any one of <1> to <12>, wherein the biological substance-immobilizing compound has a linker portion that connects the hydrophobic chain and the hydrophilic chain.
<14> A binding inhibitor that inhibits the bond between the biological substance and the hydrophobic chain to the side chain of the biological substance-immobilized compound branched from the linker portion; and the bond is cleaved by a photoreaction or The above <13>, wherein the hydrophobic chain has a photoreactive group whose structure changes; the binding inhibition by the binding inhibitory group is eliminated by light irradiation, and the hydrophobic chain can be bound to the biological substance. Immobilization material;
<15> The photoreactive groups are 2-nitrobenzyl skeleton, coumarin-4-ylmethyl skeleton, phenylcarbonylmethyl skeleton, 7-nitroindolinocarbonyl skeleton, azobenzene skeleton, flugide skeleton, spiropyran skeleton, spiroxazine skeleton and diarylethane skeleton. The immobilization material according to <14> above, which is a divalent group having a skeleton selected from the group consisting of;
<16> The immobilization material according to <14> or <15> above, wherein the binding inhibitor is a saturated or unsaturated hydrocarbon chain which may have a substituent;
<17> The polymer has a photodegradable group in the molecule, and the hydrogel thin film layer can be decomposed by cleaving the photodegradable group by light irradiation. The immobilization material according to any one of <16>;
<18> The photodegradable group is a 2-nitrobenzyl skeleton, a nitrophenylethyl ester skeleton, a coumarin-4-ylmethyl skeleton, a phenylcarbonylmethyl skeleton, a 7-nitroindolinocarbonyl skeleton, an azobenzene skeleton, a flugide skeleton, a spiropyran skeleton. The immobilization material according to <17> above, which is a divalent group having a skeleton selected from the group consisting of a spiroxazine skeleton and a diarylethane skeleton; The immobilization material according to any one of <1> to <18>, which is selected from the group consisting of vesicles, viruses, liposomes, and micelles.

Further, in another aspect, the present invention also relates to a base material for immobilizing a biological substance whose surface is modified with the above-mentioned immobilization material and a method for producing the same.
<20> An immobilization base material for immobilizing a biological substance having a lipid film on the surface, and the surface modified by the immobilization material according to any one of <1> to <19> above. The base material for immobilization, which is present on the base material in the order of the hydrogel thin film layer and the biomaterial immobilization compound;
<21> A method for producing a base material for immobilizing a biological substance having a surface modified by the immobilization material according to any one of <1> to <19> above, wherein the group is a hydrogel thin film layer made of a polymer. The step of modifying the entire surface of the material; and the surface of the hydrogel thin film layer is modified with a biological material-immobilized compound having a hydrophobic chain capable of interacting with the biological material and a hydrophilic chain connected to the surface of the hydrogel thin film layer. The production method; and <22> a binding inhibitor in which the biomaterial-immobilized compound inhibits the bond between the biomaterial and the hydrophobic chain; and the bond is cleaved by a photoreaction or In the case of having a photoreactive group whose structure changes, it is a step of patterning the surface so that the binding property with a biological substance is imparted only to a specific region on the substrate, which is the step of patterning the surface on the surface of the substrate. The production method according to <22> above, further comprising the step of irradiating a specific region with light to eliminate the binding inhibition due to the binding inhibitory group;
Is to provide.

In a further aspect, the present invention also relates to a method of immobilizing and recovering a target biological substance using the above-mentioned substrate for immobilizing a biological substance.
<23> A step of bringing a solution containing a predetermined target biological substance into contact with the immobilization base material according to <20> to immobilize the target biological material on the immobilization base material, and immobilization. A method for recovering a biological substance, which comprises a step of separating and recovering the target biological substance from the immobilization substrate; and <24> when the polymer has a photodegradable group in the molecule, the fixation. A step of separating and recovering only the target biological substance immobilized in the specific region from the immobilization substrate by irradiating a specific region of the chemical base material with light to decompose the hydrogel thin film layer. The recovery method according to <23> above is provided.

According to the present invention, a biological substance having a lipid film such as cells can be quickly and easily immobilized on the surface of a gel thin film in a uniform and high density, and the surface of the gel thin film is non-adhesive and weakly adherent. It has the effect of being able to be firmly immobilized on cells and the like.

In a preferred embodiment of the present invention, by using a photoresponsive compound as the biomaterial immobilization compound, cells and the like can be arranged with micrometer-order precision only at a desired position irradiated with light. Further, in the embodiment using a photodegradable hydrogel as the hydrogel thin film layer, among the cells immobilized on the surface of the gel thin film, only the cells existing at the desired position irradiated with light are refined on the order of micrometers. It is also possible to collect it at.

Since a plurality of types of cells are complicatedly arranged in a living tissue, by arranging the desired cells at a desired position on a hydrogel thin film according to the present invention, a tissue or tissue for transplantation having a function similar to that of a tissue in the living body. It is possible to build a model. In addition, by arranging various types of cells on the hydrogel thin film, it is possible to perform drug screening for many types of cell samples at once, and it is possible to increase the throughput of drug search and save the reagents required for the search. In particular, in recent years, it has been known that the diversity of individual cells has a great influence on the aggravation of diseases and the healing effect. Therefore, by constructing a one-cell array on a hydrogel thin film, it is covered. Accurate drug screening and cytodiagnosis are possible through conventional single-cell analysis. In addition, when the above-mentioned photodegradable hydrogel is used, a one-cell array is constructed on the hydrogel thin film, and after comprehensive one-cell analysis, high-speed cell sorting by light irradiation becomes possible.

FIG. 1 is a schematic view showing the overall structure of the biomaterial immobilization material of the present invention. The figure on the left shows the state before immobilization of the biological substance (cell), and the figure on the right shows the state in which the biological substance is immobilized on the surface of the biological substance immobilizing material. FIG. 2 (1) is a schematic view showing a typical structure of the biological substance-immobilized compound (B) used in the present invention. FIG. 2 (2) is a schematic diagram showing a structure of a preferred embodiment of the biological substance-immobilized compound (B) having a binding inhibitory group and a photoreactive group in the molecule. FIG. 3 is a schematic view of a base material surface-modified with the biological substance-immobilizing material of the present invention and a conceptual diagram of cell immobilization / recovery. FIG. 3 (i) is a representative example, and FIGS. 3 (ii) and (iii) show preferred embodiments for selective immobilization and selective recovery of cells. FIG. 4 is a microscopic image of cells immobilized in a spot shape using an aqueous PEG glycol solution on a PEG gel thin film in which fluorescent beads are dispersed. (A) Bright-field image of immobilized EGFP-BaF3 cells; (b) Green fluorescent image of immobilized EGFP-BaF3 cells; (c) Green when focused on the surface of the glass substrate under the gel thin film Fluorescence image; (d) Green fluorescence image when focused 15 μm above the surface of the glass substrate; (e) Green fluorescence image when focused 25 μm above the surface of the glass substrate. The concentration of PEG lipid used for PEG lipid modification was 20 μM, and the scale bar was 20 μm. FIG. 5 is a microscopic image of cells immobilized in spots on a PEG gel thin film using PEG lipid aqueous solutions of various concentrations. The concentrations of the PEG lipid aqueous solution are (a) 50 μM; (b) 100 μM; (c) 500 μM, respectively. FIG. 6 is a microscopic image of cells immobilized in a spot shape using an aqueous PEG lipid solution on a gelatin gel thin film in which fluorescent beads are dispersed. (A) Bright-field image of immobilized EGFP-BaF3 cells; (b) Green fluorescent image of immobilized EGFP-BaF3 cells; (c) Green when focused on the surface of the glass substrate under the gel thin film. Fluorescent image; (d) A green fluorescent image when focused 15 μm above the surface of the glass substrate. The concentration of PEG lipid used for PEG lipid modification was 20 μM, and the scale bar was 20 μm. FIG. 7 is an image showing immobilization of KO-BaF3 cells via PEG lipid on a gel thin film stained with a green fluorescent dye in a microchannel. (A) Bright-field microscope image of the surface of the gel thin film modified with PEG lipid concentration of 0 μM (top), 10 μM (middle), 100 μM (bottom); (b) Similar green fluorescence image; (c) Similar red fluorescence image; (D) This is a superposed image of a fluorescence image taken at a high magnification by shifting the focus upward from the bottom surface of the flow path modified with a PEG lipid concentration of 10 μM. FIG. 8 is an image showing patterning of KO-BaF3 cells via a photoactivated PEG lipid on a gel thin film. (A) Bright-field microscope image of the surface of the gel thin film; (b) Enlarged view of the inside of the red frame in (a); (c) The boundary region between the light-irradiated region and the non-irradiated region is directed upward from the bottom surface of the flow path. This is a superposed image of fluorescent images taken at high magnification with a different focus (the upper half of the frame is the light irradiation area, and the lower half is the non-irradiation area). FIG. 9 shows the results of PEG lipid-mediated immobilization of KO-BaF3 cells on a photosoluble gel thin film and selective cell desorption by light irradiation. (A) Structural formula of the photodegradable gel material used to prepare the photosoluble hydrogel; (b) Green fluorescence image of the gel surface before and after light irradiation; (c) Similar red fluorescence image; (d) Light after light irradiation Fluorescent image (the upper half of the frame is the light irradiation area and the lower half is the non-irradiation area) obtained by continuously capturing the boundary area between the irradiated area and the non-irradiated area while shifting the observation focus upward; (e) (d) images It is a three-dimensional image constructed from. FIG. 10 is an image showing the construction of a 1-cell array of KO-BaF3 cells mediated by photoactivated PEG lipids on a gel thin film stained with a green fluorescent dye. (A) Bright-field microscope image on the surface of the gel thin film; (b) Similar green fluorescence microscope image; (c) Similar red fluorescence microscope image. The scale bar is 500 μm. FIG. 11 is an image showing the construction of a 1-cell array of colorectal cancer B16-F10 cells via a photoactivated PEG lipid on a gelatin and PEG composite gel thin film and the results of culturing on the array. (A) Bright-field microscope image of the gel thin film surface before culturing; (b) Enlarged view in the red frame of (a); (c) Bright-field microscope image of the gel thin film surface after culturing; (d) (c) ) Is an enlarged view of the inside of the red frame. The scale bar is 100 μm. FIG. 12 shows KO-BaF3 cells and EGFP-BaF3 cells immobilized on a photosoluble gel thin film stained with a green fluorescent dye via PEG lipids, selectively by irradiation with a 1-cell size light spot. It shows the result of removing only BaF3 cells. (A) A superposed image of a green fluorescence microscope image and a red fluorescence microscope image of the surface of a gel thin film before light irradiation; (b) A similar superposed image after cleaning the surface after light irradiation. The scale bar is 100 μm.

Hereinafter, embodiments of the present invention will be described. The scope of the present invention is not limited to these explanations, and other than the following examples, the scope of the present invention can be appropriately modified and implemented as long as the gist of the present invention is not impaired.

1. 1. Biomaterial immobilization material The biomaterial immobilization material of the present invention is a material for immobilizing a biomaterial having a lipid membrane on a substrate, and as shown in FIG.
It is characterized by having a hydrogel thin film layer made of (A) polymer and (B) a modified biological substance-immobilized compound on the hydrogel thin film layer. The biological substance-immobilized compound (B) is
(A) A hydrophobic chain having a function of interacting with a biological substance and binding to the biological substance, and
(B) It has a hydrophilic chain connected to the surface of the hydrogel thin film layer. Here, the reactive group X at the end of the hydrophilic chain (b) and the reactive group Y at the end of the polymer constituting the hydrogel thin film layer (A) are covalently bonded to form the biological substance-immobilized compound (B). Is linked to the surface of the hydrogel thin film layer (A). Then, as shown in FIG. 1 (right figure), the water-repellent chain (a) can be bound and captured with a biological substance by an interaction such as a hydrophobic interaction, whereby the surface of the immobilized material can be specified. Biomaterials are immobilized in the area.

As the biological substance to be immobilized in the present invention, a structure having a lipid membrane can be widely targeted. By linking the lipid membrane with the hydrophobic chain of the biological substance-immobilized compound (B) by interaction, the biological substance can be immobilized on the surface of the material. Here, the "lipid membrane" is a membrane-like lipid. In the present specification, the term "lipid" refers to a group of substances that are poorly soluble in water and easily soluble in organic solvents. Lipids typically include long-chain fatty acids and derivatives or analogs thereof, but herein also include organic compounds such as steroids, carotenoids, terpenoids, isoprenoids, fat-soluble vitamins. Examples of lipids include simple lipids (esters of fatty acids and alcohols, which are also called neutral lipids. For example, fats and oils (triacylglycerols), waxes (waxes, fatty acid esters of higher alcohols), sterol esters, fatty acid esters of vitamins, etc. ); Complex lipids (compounds having an ester bond or an amide bond and having polar groups such as phosphoric acid, sugar, sulfuric acid, and amine in addition to fatty acids and alcohols. For example, phospholipids (glycerophospholipids and sphingos). Phosphorlipids, etc.), glycolipids (glyceroglycolipids, sphingolipids, etc.), lipoproteins, sulfolipids, etc.); Induced lipids (simple lipids and compounds produced by hydrolysis of complex lipids, which are fat-soluble) (Includes, but is not limited to, fatty acids, higher alcohols, fat-soluble vitamins, steroids, hydrocarbons, etc.).

Examples of biological substances having such a lipid membrane include cells, organelles, vesicles, viruses, liposomes, micelles, and the like. Here, the "cell" can include animal cells, plant cells, insect cells, prokaryotic cells, fungal cells, etc., and generally does not adhere or extend to the surface of a carrier such as a culture instrument, but is suspended or precipitated. Proliferating "floating cells" (for example, blood cell cells) and "adherent cells" that adhere to and extend to the surface of the carrier are dispersed from the carrier with an appropriate dispersant such as EDTA-trypsin or dispase, and temporarily suspended. Includes cells (eg, fibroblasts detached from the carrier with EDTA solution) and cells attached to the carrier. In addition, living organisms having a phospholipid bilayer on the surface such as liposomes, exosomes, bacteria, viruses, organelles, and plant cells (protoplasts) from which the cell wall has been removed are also included. Further, according to the immobilization material of the present invention, in addition to these, a substance having a lipid such as lipid-coated particles can be immobilized.

1-1. Biomaterial-immobilized compound (B)
First, among the elements constituting the biological substance-immobilizing material of the present invention, the biological substance-immobilizing compound (B) will be described. As shown in FIG. 2 (1), the biological substance-immobilized compound (B) has a structure in which a hydrophobic chain (a) and a hydrophilic chain (b) are linked, and these have a linker portion (c). ) May be connected.

The hydrophobic chain (a) of the biomaterial-immobilized compound is a site for capturing the biomaterial by binding to the target biomaterial by interaction. 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 portion in a cell membrane or the like, which is a lipid bilayer membrane.

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

In the present specification, when it is defined as "may have a substituent", the type of the substituent, the position of the substituent, and the number of the substituents are not particularly limited, and two or more substituents are used. If they have, 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 carboxyl group, a halogen atom, a sulfo group, an amino group, an alkoxycarbonyl group, an oxo group and the like. Further substituents may be present in these substituents.

The hydrophilic chain (b) is preferably composed of a hydrophilic polymer. Examples of such hydrophilic polymers include polysaccharides such as polyalkylene glycol, polyvinyl alcohol, polyacrylic acid, polypeptide, polyacrylamide, and dextran, or polymers and copolymers of glycolic acid derivatives, lactic acid derivatives, and p-dioxane derivatives. Etc. can be used. As the polyalkylene glycol, a polymer having an oxyalkylene unit having 2 to 4 carbon atoms is preferable, and one having an average polymerization number in the range of 2 to 500 (preferably 45 to 500) can be used. The hydrophilic polymer is preferably a biocompatible polymer, more preferably polyethylene glycol (PEG). The polyethylene glycol preferably has an average molecular weight of 2000 or more. The hydrophilic chain (b) may further have an arbitrary substituent.

As described above, for the purpose of linking the biological substance-immobilized compound (B) to the surface of the hydrogel thin film layer (A), the biological hydrogel thin film layer is formed in the molecule of the hydrophilic chain (b), preferably at the end thereof. It has a reactive group X that can be covalently bonded to the reactive group Y at the end of the polymer constituting the above. Preferably, the reactive group X is not particularly limited as long as it is a functional group that can covalently bond with the reactive group Y, and a functional group known in relation to the reactive group Y that can be introduced into the polymer described later can be used. , The reaction group X and the reaction group Y are preferably a combination capable of forming a covalent bond in a hydrophilic solvent environment such as water.

For example, the combination of the reactive group X and the reactive group Y includes an amino group and an N-hydroxy-succinimidyl (NHS) group; an amino group and a sulfosuccinimidyl group; an amino group and a nitrophenyl ester group; an azido group and an alkin group. (Cyclic addition reaction); Azido group and dibenzocyclooctyn group (Cyclic addition reaction); Thiol group and Maleimide group (Michael addition reaction); Thiol group and iodoacetamide group; Thiol group and vinyl sulfone group; Group; ketone group and hydrazine group; aldehyde group and aminooxy group; ketone group and aminooxy group and the like can be exemplified. In these combinations, the reaction group X and the reaction group Y can be interchanged with each other.

In a preferred embodiment, the reactive group X is a nucleophilic functional group and the reactive group Y is a nucleophilic functional group; or the reactive group X is a nucleophilic functional group and the reactive group Y is a nucleophilic functional group. Combinations that are functional groups can be used.

Examples of such a nucleophilic functional group include a thiol group (-SH) and an amino group, and those skilled in the art can appropriately use a known nucleophilic functional group. Further, as the electrophilic functional group, an active ester group can be used. Examples of electrophobic functional groups include maleimidyl group, N-hydroxy-succinimidyl (NHS) group, sulfosuccinimidyl group, phthalimidyl group, imidazolyl group, acryloyl group, -CO ₂ PhNO ₂ (Ph is o-). , M-, or p-phenylene group), nitrophenyl group and the like, and other known active ester groups can be appropriately used by those skilled in the art.

Preferably, the nucleophilic functional group is an amino group and the electrophilic functional group is an N-hydroxy-succinimidyl (NHS) group. Typically, the reactive group X in the hydrophilic chain (b) can be an NHS group and the reactive group Y in the polymer of the hydrogel thin film layer (A) can be an amino group. On the contrary, the reactive group X in the hydrophilic chain (b) can be an amino group, and the reactive group Y in the polymer can be an NHS group. When the hydrogel thin film layer (A) is composed of a biomaterial such as collagen gel, the reactive group X is preferably an active ester group capable of covalently bonding with an amino group (reactive group Y) in collagen, particularly an NHS group. It is preferable to have.

The linker portion (c) that connects the hydrophobic chain (a) and the hydrophilic chain (b) is, for example, an amide bond, an ester bond, an ether bond, a thioether bond, a carbamate bond, a thiocarbamate bond, a triazole bond, a urea bond, or the like. Functional groups capable of forming a covalent bond of the ester can be used. Further, a linker structure such as an oligomer or a polymer having such a functional group can be used, or a linker having a branched chain can be used as described later. As the branched linker (c), a benzene ring having three or more reactive functional groups such as trihydric alcohol such as glycerol; benzenetriol such as hydroxyquinol and benzenetricarboxylic acid; benzenetriamine; and 4-aminosalicylic acid can be used. It can also be used.

In a preferred embodiment, as shown in FIG. 2 (2), the biomaterial-immobilized compound (B) is attached to a side chain branched from the linker portion to (d) a bond that inhibits the bond between the biomaterial and the hydrophobic chain. Inhibitors; and (e) can further have photoreactive groups whose bond is cleaved or whose structure is altered by a photoreaction. Typically, in such an embodiment, by irradiating the biomaterial-immobilized compound (B) with light such as visible light or ultraviolet light, the photoreactive group is detached from the linker portion while being linked to the binding inhibitory group; As a result, the binding inhibition by the binding inhibitory group is eliminated, and the hydrophobic chain can bind to the biological substance. That is, it is possible to impart a switching function that allows the biological substance to be immobilized and arranged only in a desired region irradiated with light.

In the present specification, the "photoreactive group" is a group whose bond in the photoreactive group is broken or its structure is changed by irradiation with light such as visible light or ultraviolet light. The photoreactive group is not particularly limited as long as it can eliminate the binding inhibition by the binding inhibitory group by photoreaction, and is, for example, 2-nitrobenzyl skeleton, nitrophenyl ethyl ester skeleton, coumarin-4-ylmethyl skeleton, phenyl. A divalent group having a carbonylmethyl skeleton or a 7-nitroindolinocarbonyl skeleton can be used. It is preferably divalent with a 2-nitrobenzyl skeleton.

［式中、右側の矢印はリンカー部への連結を示し、左側の矢印は、結合阻害基への連結を示し、Ｌ⁶はエチニレン基であるか、又は存在しない。］ In the present specification, the "divalent group having a 2-nitrobenzyl skeleton" is a divalent group having the following structure or a derivative structure thereof.

[In the formula, the arrow on the right indicates the link to the linker, the arrow on the left indicates the link to the binding inhibitor, and L ⁶ is an ethynylene group or is absent. ]

［式中、右側の矢印はリンカー部への連結を示し、左側の矢印は、結合阻害基への連結を示す。］ The following are preferable as the divalent group having a 2-nitrobenzyl skeleton.

[In the formula, the arrow on the right indicates the link to the linker, and the arrow on the left indicates the link to the binding inhibitor. ]

［式中、右側の矢印はリンカー部への連結を示し、左側の矢印は、結合阻害基への連結を示し、Ｌ⁷は、Ｃ_1-10アルキレン基であるか又は存在せず、ここで前記アルキレン基中の炭素原子は１～５個のオキソ基で置換されていてもよく、隣接する炭素原子同士が１～５個の不飽和結合で結ばれていてもよく、そして前記アルキレン基中の炭素原子のうち、１～４個の炭素原子がＮＨ、Ｎ（Ｃ_1-10アルキル）、Ｏ又はＳで置き換えられていてもよい。］ In the present specification, the "divalent group having a coumarin-4-ylmethyl skeleton" is a divalent group having the following structure or a derivative structure thereof.

[In the formula, the arrow on the right indicates the link to the linker, the arrow on the left indicates the link to the binding inhibitor, L ⁷ is a C _1-10 alkylene group or is absent, where The carbon atom in the alkylene group may be substituted with 1 to 5 oxo groups, adjacent carbon atoms may be connected by 1 to 5 unsaturated bonds, and in the alkylene group. Of the carbon atoms in the above, 1 to 4 carbon atoms _{may be replaced with NH, N (C 1-10} alkyl), O or S. ]

［式中、右側の矢印はリンカー部への連結を示し、左側の矢印は、結合阻害基への連結を示す。］ As the divalent group having a coumarin-4-ylmethyl skeleton, the following are preferable.

[In the formula, the arrow on the right indicates the link to the linker, and the arrow on the left indicates the link to the binding inhibitor. ]

［式中、右側の矢印はリンカー部への連結を示し、左側の矢印は、結合阻害基への連結を示す。］ In the present specification, the "divalent group having a phenylcarbonylmethyl skeleton" is a divalent group having the following structure or a derivative structure thereof.

[In the formula, the arrow on the right indicates the link to the linker, and the arrow on the left indicates the link to the binding inhibitor. ]

［式中、右側の矢印はリンカー部への連結を示し、左側の矢印は、結合阻害基への連結を示す。］ The following are preferable as the divalent group having a phenylcarbonylmethyl skeleton.

[In the formula, the arrow on the right indicates the link to the linker, and the arrow on the left indicates the link to the binding inhibitor. ]

［式中、右側の矢印はリンカー部への連結を示し、左側の矢印は、結合阻害基への連結を示す。］ In the present specification, the "divalent group having a 7-nitroindolinocarbonyl skeleton" is a divalent group having the following structure or a derivative structure thereof.

[In the formula, the arrow on the right indicates the link to the linker, and the arrow on the left indicates the link to the binding inhibitor. ]

［式中、右側の矢印はリンカー部への連結を示し、左側の矢印は、結合阻害基への連結を示す。］ The following are preferable as the divalent group having a 7-nitroindolinocarbonyl skeleton.

[In the formula, the arrow on the right indicates the link to the linker, and the arrow on the left indicates the link to the binding inhibitor. ]

In another form, a mechanism may be used in which the structure of the photoreactive group is changed by the photoreaction, whereby the binding inhibition by the binding inhibitory group is eliminated. The photoreactive group in that case is not particularly limited as long as it eliminates the binding inhibition by the binding inhibitory group by a photoreaction, but is divalent having, for example, an azobenzene skeleton, a flugide skeleton, a spiropirane skeleton, a spiroxazine skeleton or a diarylethene skeleton. Group of can be used.

Other specific examples of photoreactive groups include dimethoxynitrobenzyl ester group (DMNB), bromohydroxycoumarin (Bhc) group, dimethoxybenzoin group, 2-nitropiperonyloxycarbonyl (NPOC) group, and 2-nitroveratryl. Oxycarbonyl (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) ethyloxy Carbonyl (MeNPEOC) group, 9-anthrasenylmethyloxycarbonyl (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-dimethoxybenzyloxycarbonyl group, 5-bromo-7-nitroindolinyl osikicarbonyl (BNIOC) group and the like.

Further, in the present specification, the "binding inhibitory group" is a group that physically or chemically inhibits the binding of the hydrophobic chain (a) to the lipid membrane. The structure of the binding inhibitor may be the same as or different from that of the hydrophobic chain (a). The bond-inhibiting group is not particularly limited, but may be a saturated or unsaturated hydrocarbon chain which may have a substituent. An example of such a hydrocarbon chain is as described above for the hydrophobic chain (a). The binding inhibitory group (d) and the photoreactive group (e) may be directly linked, or they may be linked via an arbitrary spacer group.

As a specific example of the immobilized compound having such a binding inhibitory group (d) and a photoreactive group (e), for example, the compound disclosed in WO2016 / 158327 can be mentioned.

1-2. Hydrogel thin film layer (A)
Next, among the elements constituting the biomaterial immobilization material of the present invention, the hydrogel thin film layer (A) will be described. The hydrogel thin film layer (A) can typically be produced by forming a hydrogel thin film on the surface of the substrate as described later.

As the hydrogel thin film layer (A), a hydrogel gelled by cross-linking polymers such as hydrophilic polymers with each other can be used. In the present specification, "gel" means a broad, high-viscosity, loss-flowing polymer dispersion system, and "hydrogel" means a gel containing water as a solvent (dispersion medium). To do. A hydrogel having a network structure, particularly a three-dimensional network structure, is preferable.

The polymer used for forming the hydrogel thin film layer (A) is not particularly limited as long as it can form a hydrogel, but is typically a hydrophilic polymer, preferably a biocompatible polymer. Examples of the polymer used for the hydrogel thin film layer (A) include carbohydrate-based polymers (methyl cellulose, carboxymethyl cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, ethyl cellulose, dextrin, cyclodextrin, alginate, hyaluronic acid, chitosan, etc.); Protein-based polymers (gelatin, albumin, collagen, glycol proteins, etc.); polyester hydroxyate (biodegradable polylactide-coglycolide (PLGA), polylactic acid (PLA), polyglycolide, polyhydroxybutyrate, polycaprolactone, polyvalerolactone) , Polyphosphazene, polyorthoester, etc.); Polyanhydride; Polyethylene glycol; Polyvinylpolyhydroxyalkylmethacrylate; Polyvinylpyrrolidone; Polyvinyl alcohol; Polypolymer. Preferably, polyalkylene glycol, cellulose, cellulose derivative, hyaluronic acid, chitosan, or collagen can be used.

Among these, polyethylene glycol (PEG) is preferable as the biocompatible polymer, and polyethylene glycol (PEG) having two branches, four branches, or eight branches is particularly preferable. Since such PEG has little effect on cells, it is excellent in that cells can be recovered in a form that does not impair the original function of cells (that is, cells can be recovered while alive). The weight average molecular weight of the PEG used is preferably 500 to 100,000, more preferably 2,000 to 40,000. Here, the weight average molecular weight is a value measured by MALDI-TOF-MS.

It is known in the art that hydrogels can be obtained by cross-linking polyethylene glycols having a branched structure such as 2-branched, 4-branched, or 8-branched to each other. More specifically, a first polymer unit (PEG) having one or more nucleophilic functional groups on the side chain or terminal and a second polymer having one or more electrophilic functional groups on the side chain or terminal. Hydrogels obtained by cross-linking these polymer units with each other by mixing the units (PEG) in a solution are preferred. In this case, since unreacted nucleophilic functional groups or electrophilic functional groups derived from the polymer unit remain in the hydrogel even after the formation of the hydrogel, these are used as the reactive group Y and the hydrophilic chain (b). It is used for binding to the reactive group X in.

As such a polymer unit, polyethylene glycol having four branches (Tetra-PEG) is particularly preferable. Such a gel composed of a tetrabranched polyethylene glycol skeleton is generally known as a Tele-PEG gel, and has an electrophilic functional group such as an active ester structure and a nucleophilic functional group such as an amino group at the terminals, respectively. A network structure network is constructed by an AB type cross-end coupling reaction between two types of tetrabranched polymers having In addition, Tetra-PEG gel can be easily prepared on the spot by a simple two-component mixing of each polymer solution, and the gelation time can be controlled by adjusting the pH and ionic strength at the time of gel preparation. Is.

Here, the types of the nucleophilic functional group and the electrophilic functional group that can be used in the polymer unit are as described above for the hydrophilic chain (b). Preferably, the nucleophilic functional group is an amino group and the electrophilic functional group is an N-hydroxy-succinimidyl (NHS) group. As described above, typically, the reactive group X in the hydrophilic chain (b) can be an NHS group and the reactive group Y in the biocompatible polymer (polymer unit) can be an amino group. On the contrary, the reactive group X in the hydrophilic chain (b) can be an amino group, and the reactive group Y in the biocompatible polymer (polymer unit) can be an NHS group.

In addition to the combination of the nucleophilic functional group and the electrophilic functional group, a combination of reactive groups capable of forming a chemical bond between the polymer units in the solution can also be used. Such combinations include, for example, azido group and alkin group (cyclization addition reaction); azido group and dibenzocyclooctine group (cyclization addition reaction); thiol group and iodoacetamide group; thiol group and vinylsulfone group; aldehyde. A group and a hydrazine group; a ketone group and a hydrazine group; an aldehyde group and an aminooxy group; a ketone group and an aminooxy group and the like can also be used.

Further, in a preferred embodiment, as the hydrogel constituting the hydrogel thin film layer (A), a hydrogel (photodegradable hydrogel) formed of a polymer having a photodegradable group in the molecule can be used. In this case, since the photodegradable group is cleaved by light irradiation to decompose the hydrogel thin film layer, the desired region is irradiated with light such as visible light or ultraviolet light after immobilizing the target biological substance. Thereby, only the cells immobilized at the position can be selectively collected.

Examples of such photodegradable groups include 2-nitrobenzyl skeleton, nitrophenylethyl ester skeleton, coumarin-4-ylmethyl skeleton, phenylcarbonylmethyl skeleton, 7-nitroindolinocarbonyl skeleton, azobenzene skeleton, flugide skeleton, spiropyran skeleton, and spiro. A divalent group having a skeleton selected from the group consisting of an oxazine skeleton and a diarylethane skeleton can be used. Other specific examples of photodegradable groups include dimethoxynitrobenzyl ester group (DMNB), bromohydroxycoumarin (Bhc) group, dimethoxybenzoin group, 2-nitropiperonyloxycarbonyl (NPOC) group, and 2-nitrobella. Trilloxycarbonyl (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) ethyl Oxycarbonyl (MeNPEOC) group, 9-anthrasenylmethyloxycarbonyl (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, Examples thereof include α, α-dimethyl-3,5-dimethoxybenzyloxycarbonyl group, 5-bromo-7-nitroindolinyl ossikicarbonyl (BNIOC) group and the like.

By introducing these photodegradable groups into a polymer, a photodegradable hydrogel can be obtained, and for such an introduction method, a chemical synthesis known in the art can be used. For example, when the above-mentioned Tetra-PEG gel is used, a photodegradable hydrogel can be obtained by introducing a photodegradable group into one or more branched chains in the polymer unit of Tetra-PEG. As a specific example, Tetra-PEG having a 4-branched branched chain having the following structure can be mentioned.

2. Biomaterial Immobilization Substrate and Manufacturing Method The present invention also relates to a biomaterial immobilization substrate having a surface modified by the immobilization material. As shown in FIG. 1, the immobilization base material has a structure in which a hydrogel thin film layer (B) and a biological substance immobilization compound (A) are present on the base material in this order.

The material and shape of the base material modified by the base material for immobilizing the biological substance of the present invention are not particularly limited, and various suitable base materials can be selected according to the use and the like. For example, the shape of the base material to be modified may be substrate-like (plate-like or film-like, such as slide glass, dish, microplate, microarray substrate, etc.), or carrier (for example, particles such as beads). It may be a colloidal one), a fibrous structure, a tube, a container (eg, a test tube and a vial). Materials to be modified include glass; cement; ceramics or fine ceramics such as ceramics; polymer resins such as polyethylene terephthalate, cellulose acetate, polycarbonate, polystyrene and polymethylmethacrylate; biomaterials such as polypeptides and proteins; silicon; Examples thereof include activated carbon; porous glass; porous ceramics; porous silicon; porous activated carbon; non-woven fabric; filter paper; membrane filter; conductive material such as gold. Since an amino group, a carboxyl group, a hydroxy group, etc. are introduced into the surface of the base material to be modified, a coating treatment with a polymer such as a polycation or a treatment with a silane coupling agent having an introduction substituent on the base material surface can be performed. It may be applied, or a reactive functional group may be introduced by plasma treatment.

The biomaterial immobilization substrate having a surface modified by the immobilization material of the present invention can typically be produced by the process shown in FIG. 3 (i). That is, the method for producing a base material for immobilizing a biological substance includes the following steps.
(P) A step of modifying the entire surface of the substrate with a hydrogel thin film layer made of a polymer; and (q) having a hydrophobic chain capable of interacting with the biological substance and a hydrophilic chain linked to the surface of the hydrogel thin film layer. A step of modifying the surface of the hydrogel thin film layer with a biomaterial-immobilized compound.

The surface modification in the step (p) can be performed, for example, by applying a solution containing a polymer or a hydrogel on the surface of the substrate. For such coating, a method known in the art such as a spacer or a bar coder can be used. The solution to be applied may contain any additive such as a cross-linking agent for gelling the polymer and a pH adjuster.

Step (q) can be performed by dropping or applying a solution containing an immobilized compound having a hydrophobic chain and a hydrophilic chain to the surface of the hydrogel thin film layer formed in step (p). As described above, the immobilized compound is modified on the surface of the hydrogel thin film layer by forming a covalent bond with the reactive group Y of the hydrogel thin film layer and linking the reactive group X of the hydrophilic chain. The concentration of the immobilized compound in the solution can be in the range of 3 to 2000 μM.

In a preferred embodiment, in the method for producing a base material for immobilizing a biological substance, by using a photoresponsive immobilizing compound, only the region irradiated with light such as visible light or ultraviolet light has a binding property with the biological substance. It is a patterned surface modification that allows the targeted biomaterial to be immobilized in a specific region of the substrate surface. For example, it is possible to have a plurality of spot-type modified regions in order to immobilize one cell. The diameter of the spot to immobilize a cell can be approximately 2-30 μm, or 5-15 μm, depending on the size of the target cell. Here, the "photoresponsive immobilized compound" is a binding inhibitor that inhibits the bond between a biological substance and a hydrophobic chain, as described above; and the bond is cleaved or its structure by a photoreaction. Means an immobilized compound having a photoreactive group that changes (as described above for the bond inhibitor and the photoreactive group).

As shown in FIG. 3 (ii), the base material for immobilizing a biological substance having such a patterned surface modification is prepared by further performing the following step (r) after the above step (q). Can:
(R) A step of patterning a surface so that binding to a biological substance is imparted only to a specific region on a substrate, wherein the specific region on the surface of the substrate is irradiated with light to inhibit the binding. The step of eliminating the binding inhibition due to.

In the step (r), the wavelength of the light to be irradiated may be determined according to the type of the photoreactive group, and usually, the light having a wavelength in the range of 157 to 600 nm, preferably a wavelength in the vicinity of 250 to 450 nm is irradiated. However, when the photoreaction is carried out by multiphoton absorption, it is possible to use light having a longer wavelength than the above. As the light source, sunlight, electric lamp light such as a mercury lamp, laser light (semiconductor laser, solid-state laser, gas laser), light emission of a light emitting diode, light emission of an electroluminescent element, etc. can be used. As a method of light irradiation, the light from the light source can be uniformly irradiated to the surface of the base material through an appropriate filter as needed, or a pattern exposure of a desired shape may be performed using a so-called mask. .. Alternatively, the light may be focused using a lens or a mirror and irradiated into a fine shape. Alternatively, the focused light rays may be scanned and exposed. In the case of pattern exposure, contact exposure, which is an exposure type in which a mask (reticle) and a work (sample) are brought into contact with each other to be exposed, may be used. Further, the proximity exposure, which is a non-contact exposure method in which the gap between the mask (reticle) and the work (sample) is set to about several μm to several tens of μm and exposed, may be performed. Further, a projection exposure method (maskless exposure method) in which an image produced by a liquid crystal or a digital mirror device is projected onto the work surface may be used. The reaction temperature is not particularly limited, but is usually −78 to 200 ° C., preferably 0 to 100 ° C., and more preferably 4 to 50 ° C. when an aqueous medium containing a biological substance such as cells is present on the work. .. Irradiation energy can be set appropriately, usually from 0.001 ~ 1000J / cm ^2, preferably 0.01 ~ 100J / cm ^2.

3. 3. Method for Immobilization and Recovery of Biological Substances Further, the present invention is a cell selection technique for immobilizing target cells and selectively recovering them using a cell immobilization substrate surface-modified with a photodegradable cell immobilizing agent. Also related. The method for recovering cells of the present invention includes the following steps:
(M) A step of bringing a solution containing a predetermined target biological substance into contact with a biological substance immobilization base material and immobilizing the target biological substance on the immobilization base material.
(N) A step of separating and recovering the immobilized target biological substance from the immobilization base material.

The separation of the biological substance in the step (n) can be performed by washing the base material, and a method known in the art can be used for the conditions and the like at the time of such washing.

As a place to perform the method, a cell immobilization substrate can be installed in the microchannel. In this case, in step (n), a flux can be imparted to the surface of the base material, and the target cells separated from the base material for cell immobilization can be recovered.

When the surface of the substrate is patterned using the above-mentioned photoresponsive immobilization compound, one cell is immobilized by having a plurality of spot-type modified regions in order to immobilize one cell.・ It is also possible to collect it.

Further, a hydrogel thin film layer (photosoluble gel thin film) formed of a polymer having a photodegradable group in the molecule can be used. In this case, since the photodegradable group is cleaved by light irradiation to decompose the hydrogel thin film layer, the desired region is irradiated with light such as visible light or ultraviolet light after immobilizing the target biological substance. As a result, only the cells immobilized at the relevant position can be selectively collected (FIG. 3 (iii)).
).

Therefore, as shown in FIG. 3 (iii), the recovery method of the present invention can include the following step (n') as a preferred embodiment of the step (n):
(N') By irradiating a specific region of the immobilization base material with light to decompose the hydrogel thin film layer, only the target biological substance immobilized in the specific region can be removed from the immobilization base material. Separation / recovery process.

The light irradiation condition in this case may be determined according to the type of photodegradable group, and can be performed under the same conditions as in the above step (r). Further, the separation of the biological substance in the step (n') can be performed by washing the base material, and a method known in the art can be used for the solution conditions and the like at the time of such washing.

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

1. 1. Preparation of hydrogel thin film 4-branched PEG (molecular weight about 10000, Sunbright PTE100GS manufactured by Nichiyu Co., Ltd.) having N-hydroxysuccinimide (NHS) ester at the end and 4-branched PEG (molecular weight about 10000, day) with amino group at the end Hydrogels were prepared by mixing Sunbright PTE100PA) manufactured by Oil Co., Ltd. in an aqueous solution to a final concentration of 5 mM. A hydrogel was also prepared in the same manner by using gelatin (manufactured by Sigma) having a final concentration of 5 wt% instead of the 4-branched PEG having an amino group. At this time, in order to prepare a flat gel thin film on the glass substrate, two 25 μm-thick silicon sheets (strip-shaped, about 5 mm × 20 mm) were attached to the glass substrate in parallel at intervals of about 10 mm. By attaching and pasting a slide glass on it, a fine space with a height of 25 μm was created, and a mixed solution of hydrogel material was quickly poured into the space, and the mixture was allowed to stand for 15 minutes or more. A small amount of fluorescent beads having a diameter of about 1 μm was suspended in this mixed solution in advance, and gelation was confirmed by observing the Brownian motion of the fluorescent beads with a fluorescence microscope (gel formation when Brownian motion stopped). ).

In order to prepare a thinner and more uniform gel thin film on the entire surface of the base material, a method using a bar coater was also performed. After reacting the above mixture of gel materials at 30 ° C for 9 minutes (reacting for 10 minutes or more, gelation occurs before the aqueous solution is expanded. On the other hand, if it is not reacted to some extent, the viscosity is insufficient and the surface is flat. It was dropped on the edge of the glass substrate and stretched with a bar coater (manufactured by AS ONE, for a 1.5 μm thin film). Then, it was allowed to stand for about 20 minutes at room temperature in a saturated steam environment.

2. Modification of Immobilization Material on Gel Thin Film and Immobilization of Cells A PEG lipid (Sunbright OE-040CS manufactured by NOF CORPORATION) having the following structure as an immobilization compound was used in the above 1. The PEG lipid was modified on the hydrogel thin film by reacting with the amino group on the hydrogel thin film prepared in 1. The PEG lipid used is a compound having a lipid chain as a hydrophobic chain, a polyethylene glycol (PEG) chain as a hydrophilic chain, and an NHS ester group capable of reacting with an NH group on the surface of a gel thin film at the end of the hydrophilic chain. Is.

After preparing a gel thin film using the above silicon spacer, remove the slide glass, immediately drop 5 μl of a 20-500 μM PEG lipid aqueous solution onto the exposed surface, and drop 3 at 37 ° C in a saturated steam environment. Reacted for time. The surface of the gel thin film was then washed with phosphate buffer (PBS) to remove unreacted PEG lipids. A suspension of mouse proB cell BaF3 strain (EGFP-BaF3 cells) expressing green fluorescent protein (EGFP) was added dropwise to the surface of this gel thin film, and the mixture was allowed to stand for 15 minutes and then removed. Furthermore, after removing non-specifically adsorbed cells by washing the surface of the gel thin film with PBS, the surface of the gel thin film was observed with a fluorescence microscope. As a result, cells were immobilized only in the region modified by dropping PEG lipid (FIGS. 4a, 4b). In addition, when the observation was made by gradually raising the observation focus from the glass surface at a larger magnification, an image in which the fluorescent beads attached to the glass were focused was seen on the glass surface (Fig. 4c), which was 15 μm higher than the glass surface. Above, an image focusing on the fluorescent beads dispersed in the gel was seen (Fig. 4d), and an image focusing on the bottom surface of EGFP-BaF3 cells was seen 25 μm above the glass surface (Fig. 4e). From this, it was confirmed that the cells were immobilized on the gel thin film having a thickness of about 25 μm via the PEG lipid.

In addition, when the gel thin film when the concentration at the time of PEG lipid modification was 50, 100, 500 μM was also observed, cell fixation was observed only in the region where the PEG lipid modification was applied, and the fixed cells were similarly observed. There was no significant difference in density (Figs. 5a-c).

On the other hand, after preparing the gel thin film, the slide glass was removed and left in a clean bench for 30 minutes or more, and after the gel thin film was dried, the same PEG lipid modification and cell fixation were attempted, and most of the cells were fixed. There wasn't. When the surface of the dried gel was observed, the polymer of the gel material formed a fibrous aggregate having a thickness of several tens of μm. Moreover, the thick fibrous aggregate did not dissolve even after being immersed in PBS again and swollen. From the above results, it is considered that when the gel material aggregates by drying after preparing the gel thin film, the PEG lipid cannot be sufficiently modified on the surface of the gel thin film and the cells are not fixed. From this, it was found that when a gel thin film with a thickness of about 25 μm is used, a gel thin film in which cells are densely fixed can be prepared by modifying the gel with a PEG lipid concentration of 20 to 500 μM before the gel dries. It was.

A similar experiment was also performed on a hydrogel thin film made of 4-branched PEG with NHS ester and gelatin, and similarly, cell fixation was observed only in the PEG lipid-modified region (similarly). Figures 6a and 6a, b). Similar to the above, when the observation focus was gradually raised from the glass surface, in the case of gelatin gel, an image in which the bottom surface of the EGFP-BaF3 cells was focused 15 μm above the glass surface (Fig. 6c) was observed. It was seen (Fig. 6d). From this, it was shown that when gelatin was used as the material, the gel contracted a little, but there was no effect on the fixation of the cells, and the cells could be immobilized on the gel thin film by this method. In this way, cells could be fixed at high density on the hydrogel thin film by this method even if the hydrogel materials and properties were different.

3. 3. Immobilization of cells on the gel thin film in the microchannel Observation and analysis of cells in the microchannel can reduce the consumption of expensive media and chemicals, can uniformly wash the cell culture surface, and can be operated. It is inexpensive and has good reproducibility because it promotes simplification and automation. Therefore, a commercially available microchamber (sticky-slide VI 0.4 manufactured by ibidi) was placed on a gel thin film prepared on a slide glass using a bar coater as described above to prepare a flow path. At this time, the gel was prepared by mixing the above two types of 4-branched PEG materials (final concentration 5 mM), and a small amount of fluorescein-5-NHS ester (final concentration 50 μM) was simultaneously observed so that the gel could be observed with a fluorescence microscope. mixed. A 10 to 100 μM PEG lipid aqueous solution was injected into this microchannel and reacted at 37 ° C. for 3 hours. Then, in order to remove the unreacted PEG lipid, PBS was flowed through the flow path to wash the gel surface. In order to block the excess amino groups on the gel surface, a 5 mM sulfo-NHS acetate aqueous solution was injected into the flow path and reacted at 37 ° C. for 3 hours, and PBS was flowed to wash the gel surface again. Then, a suspension of BaF3 cells (KO-BaF3 cells) expressing the red fluorescent protein (wedge orange: KO) was injected into the flow path, allowed to stand for 10 minutes, and then removed. Furthermore, PBS was injected 5 times to wash the surface of the gel thin film to remove non-specifically adsorbed cells, and then the surface of the gel thin film was observed with a confocal laser scanning microscope.

As a result, it was confirmed from the bright-field image and the red fluorescence image that the cells were densely and uniformly fixed over the entire flow path only on the gel surface of the flow path modified with PEG lipid (Fig. 7a). ~ C). In addition, when the flow path modified with 10 μM PEG lipid was further magnified and the observation focus was raised by about 2 μm from the glass surface, a gel having green fluorescence with a thickness of about 4 to 8 μm was observed. After the thin film was observed, cells having red fluorescence having a thickness of about 8 to 12 μm were observed on the thin film (Fig. 7d). From the above, it was confirmed that cells can be rapidly, uniformly, and densely fixed to a gel thin film thin enough not to leak from the gel layer even when covered with a microchamber.

4. Photoarrangement of cells on a gel thin film (example using photoactivated PEG lipid)
Above 3. The surface of the gel thin film prepared in the microchannel by the above method was modified with a photoactivated PEG lipid (compound of formula (Ic) described in WO2016 / 158327) and irradiated with light on the gel surface. The cells were placed only in. A 100 μM photoactivated PEG lipid aqueous solution was injected into the microchannel on the gel thin film prepared in the same manner as above, and the mixture was reacted at 37 ° C. for 3 hours. Then, it was similarly blocked with a 5 mM sulfo-NHS acetate aqueous solution. This microchannel was placed on a photomask (line pattern at 400 μm intervals) and irradiated with ^{light of 360 nm from below with a xenon lamp at 1.5 J / cm 2.} Then, in the same manner as above, the KO-BaF3 cell suspension was injected into the flow path and fixed on the gel surface. The surface of the gel thin film was washed by injecting PBS 5 times to remove non-specifically adsorbed cells, and then the surface of the gel thin film was observed with a confocal laser scanning microscope.
As a result, a line pattern of cells was observed on the gel surface, and it was confirmed that the cells were arranged only in the light irradiation region (FIGS. 8a and 8b). In addition, when the boundary between the light-irradiated region and the non-irradiated region was defocused upward from the bottom surface of the flow path and imaged at high magnification, red cells appeared on the green gel thin film depending on the presence or absence of light irradiation. It was confirmed that they were arranged (Fig. 8c). This result shows that according to the present invention, cells can be arranged at a desired position on the gel thin film.

5. Desorption of cells from the photosoluble gel thin film (example using a photosoluble hydrogel thin film)
Above 3. A photosoluble gel thin film was prepared in the microchannel in the same manner as in the above. At this time, instead of the 4-branched PEG having an NHS ester at the terminal, a 4-branched PEG having an NHS ester at the terminal (Fig. 9a) is used via a photodegradable linker, and a 4-branched PEG having an amino group at the terminal is used. It was prepared by mixing a small amount of fluorescein-5-NHS ester. PEG lipids were modified on this photosoluble gel thin film in exactly the same manner as above, blocked with sulfo-NHS acetate, and then KO-BaF3 cells were immobilized. After that, the cells immobilized on the gel surface were irradiated with line pattern light (5 J / cm ² ) using a photomask in the same manner as above, and the gel surface was washed with PBS to obtain the light irradiation area. An operation was performed to selectively collect only cells from the bottom surface of the flow path.

As a result, the cells that were uniformly fixed on the entire gel surface before the light irradiation were removed from the light irradiation area as the gel was dissolved by the light irradiation, which is the pattern of the green fluorescence of the gel and the red fluorescence of the cells. It was confirmed from (Fig. 9b, c). Similar to the above experiment, when the light-irradiated area and the non-irradiated area were enlarged and the observation focus was shifted upward for continuous imaging, the cells were fixed on the gel thin film in the non-irradiated area. It was confirmed that the gel disappeared in the light-irradiated region, and almost all the cells disappeared at the same time (Fig. 9d). This result shows that according to the present invention, floating cells can be rapidly and easily immobilized on a photosoluble gel thin film, and only cells at a desired position among the immobilized cells can be separated and recovered by light irradiation. It is a thing.

6. Construction and culture of a 1-cell array on a gel thin film 4. A gel thin film modified with a photoactivated PEG lipid was prepared in the microchannel in the same manner as in the above. This microchannel was placed on a photomask (a pattern in which circular light transmitting regions with a diameter of 22 μm were arranged in a grid pattern at intervals of 100 μm), and light of 360 nm was irradiated ^{from below at 1.5 J / cm 2 with a xenon lamp.} .. Then, in the same manner as above, the KO-BaF3 cell suspension was injected into the flow path and fixed on the gel surface. The surface of the gel thin film was washed by injecting PBS 5 times to remove non-specifically adsorbed cells, and then the surface of the gel thin film was observed with a confocal laser scanning microscope. As a result, as shown in FIG. 10, it was found that a 1-cell array of KO-BaF3 cells could be constructed on a gel thin film.

Furthermore, a 1-cell array was constructed using B16-F10 cells of colorectal cancer, and the cells were cultured on the array. Above 4. A gel thin film was prepared in the microchannel using 4-branched PEG having NHS ester and gelatin in the same manner as in the above method, and modified by injecting 10 μM and 100 μM photoactivated PEG lipid aqueous solutions. By the above method, a circular 360 nm light pattern with a diameter of 24 μm (a grid pattern at 100 μm intervals) was irradiated at ^{1.5 J / cm 2.} Then, the B16-F10 cell suspension was injected into the flow path in the same manner as above and fixed on the gel surface. The surface of the gel thin film was washed by injecting PBS 5 times to remove non-specifically adsorbed cells, and then the surface of the gel thin film was observed with a confocal laser scanning microscope. Furthermore, RPMI1640 medium was injected 5 times to replace the flow medium with a culture medium, and after culturing in a saturated steam environment at a temperature of 37 ° C. and 5% carbon dioxide for 15 hours, a gel thin film was again subjected to a confocal laser scanning microscope. The surface of was observed. As a result, on the surface of the gel thin film modified with a 10 μM photoactivated PEG lipid aqueous solution, cell elongation could be confirmed after culturing, as shown in FIG. On the other hand, no cell elongation was confirmed on the surface of the gel thin film modified with 100 μM. In this way, by modifying the photoactivated PEG lipid at a thin concentration on the gel thin film prepared using cell-adherent gelatin, a single cell array of adherent cells can be cultured in an environment close to that of a living body and adhered. It was shown that analysis in the adherent and extended state of cells is possible.

7. Light recovery at the 1-cell level using a gel thin film 5. Two types of different fluorescence are prepared by preparing a photosoluble gel thin film modified with PEG lipid in a microchannel and injecting a mixed suspension of KO-BaF3 cells and EGFP-BaF3 cells in the same manner as in Cells were immobilized. The surface of the gel thin film was observed with a confocal laser scanning microscope, and only the place where the KO-BaF3 cells emitting red fluorescence were immobilized was irradiated with a 1-cell size 405 nm laser beam for 160 ms. By washing the gel surface with PBS three times, only the light-irradiated KO-BaF3 cells were selectively recovered from the gel surface. As a result, as shown in FIG. 12, it was demonstrated that only KO-BaF3 cells could be selectively light-recovered at the single cell level by light irradiation, and high-speed sorting was possible.

Claims (24)

An immobilization material for immobilizing a biological substance having a lipid membrane on a substrate.
It has a hydrogel thin film layer made of a polymer and a structure in which a biological substance-immobilized compound is modified on the hydrogel thin film layer.
The biomaterial-immobilized compound has a hydrophobic chain capable of interacting with the biomaterial and a hydrophilic chain linked to the surface of the hydrogel thin film layer.
At the end of the hydrophilic chain, a reactive group X that can be covalently bonded to the surface of the hydrogel thin film layer is provided.
The polymer has a reactive group Y in its molecule that forms a covalent bond with the reactive group X.
The immobilization material. The immobilization material according to claim 1, wherein the hydrophilic chain contains a polyalkylene glycol. The immobilization material according to claim 1 or 2, wherein the hydrophobic chain is a saturated or unsaturated hydrocarbon chain which may have a substituent. The combination of the reactive group X and the reactive group Y is an amino group and an N-hydroxy-succinimidyl (NHS) group; an amino group and a sulfosuccinimidyl group; an amino group and a nitrophenyl ester group; an azido group and an alkin group; Azido group and dibenzocyclooctine group; thiol group and maleimide group; thiol group and iodoacetamide group; thiol group and vinylsulfone group; aldehyde group and hydrazine group; ketone group and hydrazine group; aldehyde group and aminooxy group; ketone group The immobilization material according to any one of claims 1 to 3, which is selected from the group consisting of aminooxy groups. The immobilized material according to any one of claims 1 to 3, wherein the reactive group X is a nucleophilic functional group and the reactive group Y is an electrophilic functional group. The immobilization material according to any one of claims 1 to 3, wherein the reactive group X is an electrophilic functional group and the reactive group Y is a nucleophilic functional group. The polymer according to any one of claims 1 to 6, wherein the polymer is a polyalkylene glycol, a carbohydrate-based polymer, a protein-based polymer, a polyester hydroxy acid, a polyanhydride, a polyvinylpolyhydroxyalkylmethacrylate, a polyvinylpyrrolidone, a polyvinyl alcohol, or a polyamide. The immobilized material described. The immobilization material according to any one of claims 1 to 7, wherein the polymer is polyethylene glycol (PEG) having two branches, four branches, or eight branches. The polyethylene glycol comprises a first polymer unit having one or more nucleophilic functional groups in the side chain or terminal and a second polymer unit having one or more nucleophilic functional groups in the side chain or terminal; The immobilization material according to claim 8, wherein the polymer units are crosslinked with each other to form a hydrogel. The nucleophilic functional group is selected from the group consisting of a thiol group and an amino group; the electrophilic functional group is a maleimidyl group, an N-hydroxy-succinimidyl (NHS) group, a sulfosuccinimidyl group, a phthalimidyl group. The immobilization material according to any one of claims 5 to 9, selected from the group consisting of a group, an imidazolyl group, an acryloyl group, -CO _{2 PhNO 2, and a nitrophenyl group.} The polymer is polyethylene glycol with four branches (Terra-PEG); the nucleophilic functional group is an amino group; the electrophilic functional group is an N-hydroxy-succinimidyl (NHS) group. , The immobilization material according to any one of claims 5 to 10. The immobilization material according to claim 11, wherein the reactive group X is an N-hydroxy-succinimidyl (NHS) group and the reactive group Y is an amino group. The immobilization material according to any one of claims 1 to 12, wherein the biomaterial immobilization compound has a linker portion that connects the hydrophobic chain and the hydrophilic chain. A binding inhibitor that inhibits the bond between the biological substance and the hydrophobic chain; and the bond is cleaved or its structure is formed in the side chain of the biological substance-immobilized compound branching from the linker portion. With varying photoreactive groups
The immobilization material according to claim 13, wherein the binding inhibition by the binding inhibitory group is eliminated by light irradiation, and the hydrophobic chain can be bound to the biological substance. The group in which the photoreactive group consists of a 2-nitrobenzyl skeleton, a coumarin-4-ylmethyl skeleton, a phenylcarbonylmethyl skeleton, a 7-nitroindolinocarbonyl skeleton, an azobenzene skeleton, a flugide skeleton, a spiropyran skeleton, a spiroxazine skeleton and a diarylethene skeleton. The immobilized material according to claim 14, which is a divalent group having a skeleton selected from. The immobilized material according to claim 14 or 15, wherein the binding inhibitor is a saturated or unsaturated hydrocarbon chain which may have a substituent. The polymer has a photodegradable group in the molecule and
The immobilization material according to any one of claims 1 to 16, wherein the hydrogel thin film layer can be decomposed by cleaving the photodegradable group by light irradiation. The photodegradable groups are 2-nitrobenzyl skeleton, nitrophenylethyl ester skeleton, coumarin-4-ylmethyl skeleton, phenylcarbonylmethyl skeleton, 7-nitroindolinocarbonyl skeleton, azobenzene skeleton, flugide skeleton, spiropyran skeleton, spirooxazine. The immobilized material according to claim 17, which is a divalent group having a skeleton selected from the group consisting of a skeleton and a diarylethane skeleton. The immobilization material according to any one of claims 1 to 18, wherein the biological substance is selected from the group consisting of cells, organelles, vesicles, viruses, liposomes, and micelles. An immobilization base material for immobilizing a biological substance having a lipid membrane on the surface.
For immobilization, which has a surface modified by the immobilization material according to any one of claims 1 to 19; is present on the substrate in the order of the hydrogel thin film layer and the biomaterial immobilization compound. Base material. A method for producing a base material for immobilizing a biological substance, which has a surface modified by the immobilizing material according to any one of claims 1 to 19.
A step of modifying the entire surface of a substrate with a hydrogel thin film layer made of a polymer; and a biomaterial-immobilized compound having a hydrophobic chain capable of interacting with the biomaterial and a hydrophilic chain linked to the surface of the hydrogel thin film layer. The production method comprising the step of modifying the surface of the hydrogel thin film layer. When the biomaterial-immobilized compound has a binding inhibitor that inhibits the bond between the biomaterial and the hydrophobic chain; and a photoreactive group in which the bond is cleaved or its structure is changed by a photoreaction. ,
A step of patterning a surface so that binding to a biological substance is imparted only to a specific region on the substrate, wherein the specific region on the surface of the substrate is irradiated with light and bound by the binding inhibitor. The production method according to claim 21, further comprising the step of eliminating the inhibition. A step of bringing a solution containing a predetermined target biological substance into contact with the immobilization base material according to claim 20 to immobilize the target biological material on the immobilization base material, and the immobilized target biological material. A method for recovering a biological substance, which comprises a step of separating and recovering from the immobilization base material. When the polymer has a photodegradable group in the molecule,
By irradiating a specific region of the immobilization base material with light to decompose the hydrogel thin film layer, only the target biological substance immobilized in the specific region is separated and recovered from the immobilization base material. The collection method according to claim 23, which comprises the step of performing.

Images