Biomaterial immobilization materials

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

[Technical Field]

[0001] The present invention relates to a material for quickly and simply immobilizing biological materials having lipid membranes, a cell immobilization substrate having a surface modified with the material, and a cell recovery method using the substrate. [Background technology]

[0002] It is known that in vivo, most cells adhere and spread on an extracellular matrix (ECM) to perform their normal functions, forming three-dimensional tissues supported by the periphery of the matrix. This extracellular matrix is ​​a hydrogel composed of fibrous proteins and polysaccharides, such as collagen and proteoglycans, containing water. Its primary function is as a scaffold for cells. For example, when hepatocytes are cultured ex vivo, they lose their function when directly attached to a culture substrate. However, many studies have shown that cells can maintain their function for long periods of time on substrates coated with an ECM gel (see, for example, Non-Patent Document 1). Furthermore, it has been reported that cell function is controlled by the hardness of the ECM, the liquid components contained within it, and the properties of the molecules they present. It has also been reported that certain ECM gels, such as matrix gels that promote the formation of tubular structures in vascular endothelial cells, are essential for cell function (see, for example, Non-Patent Document 2). For this reason, cells are seeded on ECM gels and cultured, assayed, and organized for drug discovery and toxicity testing based on in vivo cell function, as well as for regenerative medicine utilizing in vivo cell function.

[0003] Meanwhile, in a wide range of research fields, including 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 the ability to sustainedly release encapsulated molecules, and have been applied to a technology that simultaneously releases a variety of drugs arrayed within the gel and comprehensively analyzes their effects on cells cultured on the gel (Non-Patent Document 3). Other reported technologies include a sensing technology for cell secretions by modifying polymer fibers in the gel with fluorescent sensor molecules (Non-Patent Document 4), and a technology for selectively recovering cells adhered to a photolytic synthetic hydrogel by irradiating it with light (Non-Patent Document 5). Thus, the technology of culturing and assaying cells on synthetic polymer gels is also widely applied.

[0004] Given this background, there is a need for a technology that allows for the rapid and simple seeding of cells onto hydrogel surfaces at high density. However, conventional methods for seeding cells onto hydrogels generally involve placing a cell suspension on the surface of the hydrogel and allowing the cells to spontaneously adhere to the gel material. However, this method is only applicable to adherent cells, and is problematic in that it cannot be applied to some weakly adherent hepatocytes or many nonadherent blood and immune cells.

[0005] A method using antibodies has also been reported as a method for capturing suspension cells on a gel surface (Non-Patent Document 6). However, antibodies are expensive to produce, and antibodies with specific binding properties are particularly expensive. In addition, it is difficult to obtain antibodies that can strongly recognize the cell membrane surface of any cell.

[0006] Another reported method for dispensing cells onto hydrogels is to use an inkjet printer to eject cells (e.g., Non-Patent Document 7). However, when using an inkjet printer, cytotoxicity due to pressure and heat can be a problem. Furthermore, it is currently difficult to precisely eject individual cells. Even if it were possible to eject cells one by one, this process would take an enormous amount of time, making it difficult to precisely position cells at the single-cell level. Another reported technique involves incorporating magnetic beads into cells and then using fine magnets to position the cells on a hydrogel (Non-Patent Document 8). However, microfabrication of magnets is expensive, and there are limitations to the shape of the patterns that can be arranged. Furthermore, magnetic beads incorporated into cells cannot be removed after placement, raising concerns about the impact on cell assays and regenerative medicine.

[0007] Meanwhile, the inventors of the present invention have already reported a method for transferring cells placed on a substrate by pressing them onto a hydrogel (Non-Patent Document 9). However, this method requires adhesion between the cells and the hydrogel surface for transfer, which poses a problem in that it can only be applied to cells that have adhesive properties to the hydrogel. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Toshiaki Takezawa et al., Pharmaceutical Journal, 2008, 128, 51 [Non-patent document 2] Akhtar N, et al., Angiogenesis, 2002, 5, 75-80 [Non-patent document 3] ailey SN, et al., Proc. Natl. Acad. Soc. USA , 2004, 101, 16144-816149 [Non-patent document 4] Son KJ, et al., Anal. Chem., 2013, 85, 11893-11901 [Non-patent document 5] Tamura M., et al., Sci. Rep., 2015, 5, 15060 [Non-patent document 6] Son KJ, et al., Lab Chip, 2015, 15, 637-641 [Non-Patent Document 7] Nahmias Y., et al., Tissue Eng., 2005, 11, 701-708 [Non-patent document 8] Okochi M., et al., Lab Chip, 2009, 9, 3378-3384 [Non-Patent Document 9] Takano T., et al., Biotechnol. Bioeng., 2012, 109, 244-251 Summary of the Invention [Problem to be solved by the invention]

[0009] Therefore, an object of the present invention is to solve the problems of the conventional methods described above and to develop a system that can rapidly and easily immobilize biological materials such as cells on a thin hydrogel film. [Means for solving the problem]

[0010] As a result of intensive research aimed at solving the above-mentioned problems, the present inventors have found that by using a thin film of a hydrogel having reactive groups on its surface and modifying the reactive groups on the hydrogel surface with a biological substance immobilization compound capable of interacting with the biological substance, it is possible to rapidly immobilize any biological substance, such as cells, on the hydrogel surface. They also found that by using a photoreactive biological substance immobilization compound or hydrogel, cells and the like can be selectively immobilized or recovered by light irradiation. These findings led to the completion of the present invention.

[0011] That is, in one aspect, the present invention provides: <1> An immobilization material for immobilizing a biological material having a lipid membrane on a substrate, the immobilization material comprising a hydrogel thin film layer made of a polymer and a structure in which a biological material immobilization compound is modified on the hydrogel thin film layer; the biological material immobilization compound has 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 hydrophilic chain has a reactive group X at the end thereof capable of bonding to the surface of the hydrogel thin film layer by a covalent bond; and the polymer molecule has a reactive group Y that forms a covalent bond with the reactive group X. <2> The hydrophilic chain comprises polyalkylene glycol. <1> 1. The immobilization material according to claim 1 ; <3> The hydrophobic chain is a saturated or unsaturated hydrocarbon chain which may have a substituent. <1> or <2> 1. The immobilization material according to claim 1 ; <4> the combination of the reactive group X and the reactive group Y is selected from the group consisting of 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 azide group and an alkyne group; an azide group and a dibenzocyclooctyne group; a thiol group and a maleimide group; a thiol group and an iodoacetamide group; a thiol group and a vinyl sulfone group; an aldehyde group and a hydrazine group; a ketone group and a hydrazine group; an aldehyde group and an aminooxy group; or a ketone group and an aminooxy group; <1> ~ <3> 1. The immobilization material according to any one of the preceding items. <5> The reactive group X is a nucleophilic functional group, and the reactive group Y is an electrophilic functional group. <1> ~ <3> 1. The immobilization material according to any one of the preceding items. <6> The reactive group X is an electrophilic functional group, and the reactive group Y is a nucleophilic functional group. <1> ~ <3> 1. The immobilization material according to any one of the preceding items. <7> The above-mentioned polymer is a carbohydrate-based polymer, a protein-based polymer, a hydroxy acid polyester, a polyanhydride, a polyvinyl polyhydroxyalkyl methacrylate, a polyvinylpyrrolidone, a polyvinyl alcohol, or a polyamide. <1> ~ <6> 1. The immobilization material according to any one of the preceding items. <8> The polymer is polyethylene glycol (PEG) having two branches, four branches, or eight branches. <1> ~ <7> 1. The immobilization material according to any one of the preceding items. <9> The polyethylene glycol is composed of a first polymer unit having one or more nucleophilic functional groups at the side chain or at the end and a second polymer unit having one or more electrophilic functional groups at the side chain or at the end; and the polymer units are crosslinked with each other to form a hydrogel. <8> 1. The immobilization material according to claim 1 ; <10> the nucleophilic functional group is selected from the group consisting of a thiol group and an amino group; and the electrophilic functional group is selected from the group consisting of a maleimidyl group, an N-hydroxy-succinimidyl (NHS) group, a sulfosuccinimidyl group, a phthalimidyl group, an imidazoyl group, an acryloyl group, -CO2PhNO2, and a nitrophenyl group. <5> ~ <9> 1. The immobilization material according to any one of the preceding items. <11> The polymer is a 4-branched polyethylene glycol (Tetra-PEG); the nucleophilic functional group is an amino group; and the electrophilic functional group is an N-hydroxy-succinimidyl (NHS) group. <5> ~ <10> 1. The immobilization material according to any one of the preceding items. <12> The reactive group X is an N-hydroxy-succinimidyl (NHS) group, and the reactive group Y is an amino group. <11> 1. The immobilization material according to claim 1 ; <13> The biological material immobilization compound has a linker portion connecting the hydrophobic chain and the hydrophilic chain. <1> ~ <12> 1. The immobilization material according to any one of the preceding items. <14> the biological substance immobilization compound has, in a side chain branched from a linker portion, a bond-inhibiting group that inhibits the bond between the biological substance and the hydrophobic chain; and a photoreactive group whose bond is cleaved or whose structure is changed by a photoreaction; and the binding inhibition by the bond-inhibiting group is eliminated by light irradiation, so that the hydrophobic chain can bind to the biological substance. <13> 1. The immobilization material according to claim 1 ; <15> the photoreactive group is a divalent group having a skeleton selected from the group consisting of a 2-nitrobenzyl skeleton, a coumarin-4-ylmethyl skeleton, a phenylcarbonylmethyl skeleton, a 7-nitroindolinocarbonyl skeleton, an azobenzene skeleton, a fulgide skeleton, a spiropyran skeleton, a spirooxazine skeleton, and a diarylethene skeleton; <14> 1. The immobilization material according to claim 1 ; <16> The above-mentioned binding inhibitor group is a saturated or unsaturated hydrocarbon chain which may have a substituent. <14> or <15> 1. The immobilization material according to claim 1 ; <17> The polymer has a photodegradable group in the molecule, and the photodegradable group is cleaved by light irradiation, thereby decomposing the hydrogel thin film layer. <1> ~ <16> 1. The immobilization material according to any one of the preceding items. <18> the photodegradable group is a divalent group having a skeleton selected from the group consisting of a 2-nitrobenzyl skeleton, a nitrophenylethyl ester skeleton, a coumarin-4-ylmethyl skeleton, a phenylcarbonylmethyl skeleton, a 7-nitroindolinocarbonyl skeleton, an azobenzene skeleton, a fulgide skeleton, a spiropyran skeleton, a spirooxazine skeleton, and a diarylethene skeleton; <17> an immobilization material according to the present invention; and <19> The biological material is selected from the group consisting of cells, organelles, vesicles, viruses, liposomes, and micelles. <1> ~ <18> The immobilization material according to any one of the preceding claims This provides:

[0012] In another aspect, the present invention also relates to a substrate for immobilizing a biological material whose surface is modified with the above-mentioned immobilization material and a method for producing the same, more specifically, <20> An immobilization substrate for immobilizing a biological material having a lipid membrane on a surface thereof, comprising: <1> ~ <19> a substrate for immobilization having a surface modified with the immobilization material according to any one of the above items (1) to (5); and the substrate having the hydrogel thin film layer and the biological substance immobilization compound present on the substrate in this order; <21> the above <1> ~ <19> 1. A method for producing a substrate for immobilizing a biological substance, the surface of which is modified with the immobilization material according to any one of claims 1 to 9, comprising the steps of: modifying the entire surface of the substrate with a hydrogel thin film layer made of a polymer; and modifying the surface of the hydrogel thin film layer with a biological substance immobilization compound 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; and <22> When the biological substance immobilization compound has a binding-inhibiting group that inhibits binding between the biological substance and the hydrophobic chain; and a photoreactive group whose bond is cleaved or whose structure is changed by a photoreaction, the method further comprises a step of patterning the surface of the substrate so that only specific regions on the substrate are endowed with the ability to bind to the biological substance, and irradiating the specific regions on the surface of the substrate with light to eliminate the binding inhibition caused by the binding-inhibiting group. <22> 2. The method of manufacturing a semiconductor device according to claim 1 ; This provides:

[0013] In a further aspect, the present invention also relates to a method for immobilizing and recovering a target biological material using the above-mentioned substrate for immobilizing a biological material, more specifically, the method comprising: <23> the above <20> a step of contacting a solution containing a predetermined target biological substance with the immobilization substrate described in the above item 1 to immobilize the target biological substance on the immobilization substrate; and a step of separating and recovering the immobilized target biological substance from the immobilization substrate. a method for recovering biological material, comprising: <24> When the polymer has a photodegradable group in the molecule, the method further comprises a step of irradiating a specific region of the immobilization substrate with light to decompose the hydrogel thin film layer, thereby separating and recovering only the target biological substance immobilized in the specific region from the immobilization substrate. <23> The recovery method described in This provides: [Effects of the Invention]

[0014] According to the present invention, biological materials having lipid membranes, such as cells, can be immobilized on the surface of a thin gel film quickly and easily, uniformly, and at high density, and even non-adhesive or weakly adhesive cells can be firmly immobilized on the surface of a thin gel film.

[0015] In a preferred embodiment of the present invention, a photoresponsive compound is used as the biological material immobilization compound, thereby enabling cells, etc. to be positioned with micrometer-order precision only at the desired position irradiated with light. Furthermore, in an embodiment in which a photodegradable hydrogel is used as the hydrogel thin film layer, it is also possible to recover, with micrometer-order precision, only the cells present at the desired position irradiated with light from among the cells, etc. immobilized on the surface of the gel thin film.

[0016] Because biological tissues contain multiple types of cells arranged in complex locations, the present invention allows for the construction of transplantable tissues and tissue models with functions similar to those of in vivo tissues by arranging desired cells at desired locations on a hydrogel thin film. Furthermore, arranging multiple types of cells on a hydrogel thin film also enables drug screening of multiple cell samples at once, thereby enabling high-throughput drug discovery and saving on reagents required for discovery. In particular, since it has recently become clear that the diversity of individual cells has a significant impact on the severity of disease and healing outcomes, constructing a single-cell array on a hydrogel thin film enables accurate drug screening and cytological diagnosis through comprehensive single-cell analysis. Furthermore, when using the photodegradable hydrogel described above, constructing a single-cell array on a hydrogel thin film and then conducting comprehensive single-cell analysis can enable high-speed cell sorting by light irradiation. [Brief explanation of the drawings]

[0017] [Figure 1] Figure 1 is a schematic diagram showing the overall structure of the biological substance-immobilized material of the present invention. The left figure shows the state before biological substances (cells) are immobilized, and the right figure shows the state after biological substances have been immobilized on the surface of the biological substance-immobilized material. [Figure 2]Fig. 2(1) is a schematic diagram showing a typical structure of the biological substance-immobilizing compound (B) used in the present invention. Fig. 2(2) is a schematic diagram showing the structure of a preferred embodiment of the biological substance-immobilizing compound (B) further having a binding inhibitor group and a photoreactive group in the molecule. [Figure 3] Figure 3 shows a schematic diagram of a substrate surface-modified with the biosubstance-immobilizing material of the present invention and a conceptual diagram of cell immobilization and recovery. Figure 3(i) is a representative example, and Figures 3(ii) and (iii) show preferred embodiments for selective immobilization and selective recovery of cells. [Figure 4] Figure 4 shows microscopic images of cells immobilized in spots using a PEG-lipid solution on a thin PEG gel film containing dispersed fluorescent beads. (a) Bright-field image of immobilized EGFP-BaF3 cells; (b) Green fluorescent image of immobilized EGFP-BaF3 cells; (c) Green fluorescent image focused on the glass substrate surface below the thin gel film; (d) Green fluorescent image focused 15 μm above the glass substrate surface; and (e) Green fluorescent image focused 25 μm above the glass substrate surface. The PEG-lipid concentration used for PEG-lipid modification was 20 μM; the scale bar is 20 μm. [Figure 5] Figure 5 shows microscopic images of cells immobilized on a thin PEG gel film in spots using aqueous PEG-lipid solutions of various concentrations: (a) 50 μM, (b) 100 μM, and (c) 500 μM. [Figure 6] Figure 6 shows microscopic images of cells immobilized in spots using a PEG-lipid aqueous solution on a thin gelatin gel film containing dispersed fluorescent beads. (a) Bright-field image of immobilized EGFP-BaF3 cells; (b) Green fluorescent image of immobilized EGFP-BaF3 cells; (c) Green fluorescent image focused on the glass substrate surface below the thin gel film; (d) Green fluorescent image focused 15 μm above the glass substrate surface. The PEG-lipid concentration used for PEG modification was 20 μM; the scale bar is 20 μm. [Figure 7]Figure 7 shows images of KO-BaF3 cell immobilization via PEG-lipids on a thin gel membrane stained with green fluorescent dye in a microchannel. (a) Bright-field microscopy images of the surface of the thin gel membrane modified with PEG-lipid concentrations of 0 μM (top), 10 μM (middle), and 100 μM (bottom); (b) a similar green fluorescent image; (c) a similar red fluorescent image; and (d) a superimposed image of fluorescent images taken at high magnification with the focus shifted from the bottom to the top of the channel modified with PEG-lipid concentration of 10 μM. [Figure 8] Figure 8 shows images of KO-BaF3 cell patterning on a thin gel film via photoactivatable PEG-lipids. (a) Bright-field microscope image of the thin gel film surface; (b) Enlarged view of the area within the red frame in (a); (c) Superimposed fluorescent image of the boundary between the illuminated and non-illuminated regions, taken at high magnification with the focus shifted upward from the bottom of the channel (the upper half of the frame is the illuminated region, and the lower half is the non-illuminated region). [Figure 9] Figure 9 shows the results of immobilization of KO-BaF3 cells on a thin film of photolytic gel via PEG-lipids and selective cell detachment by light irradiation. (a) Structural formula of the photodegradable gel material used to prepare the photolytic hydrogel; (b) Green fluorescent images of the gel surface before and after light irradiation; (c) A similar red fluorescent image; (d) Fluorescent images taken after light irradiation, with the observation focus shifted upward at the boundary between the irradiated and non-irradiated regions (the upper half of the frame is the irradiated region, and the lower half is the non-irradiated region); (e) A three-dimensional image constructed from the images in (d). [Figure 10] Figure 10 shows images showing the construction of a single-cell array of KO-BaF3 cells via photoactivatable PEG-lipids on a thin gel film stained with green fluorescent dye. (a) Bright-field microscopy image of the thin gel film surface; (b) a similar green fluorescent microscopy image; (c) a similar red fluorescent microscopy image. Scale bar: 500 μm. [Figure 11]Figure 11 shows images of the construction of a single-cell array of colon cancer B16-F10 cells on a gelatin-PEG composite gel film via photoactivatable PEG-lipids and the results of culturing on the array. (a) Bright-field microscope image of the gel film surface before culturing; (b) Enlarged image of the area enclosed by the red frame in (a); (c) Bright-field microscope image of the gel film surface after culturing; (d) Enlarged image of the area enclosed by the red frame in (c). The scale bar is 100 μm. [Figure 12] Figure 12 shows the selective removal of KO-BaF3 cells from KO-BaF3 and EGFP-BaF3 cells immobilized via PEG lipids on a photolytic gel film stained with a green fluorescent dye by irradiating it with a single-cell-sized light spot. (a) Green and red fluorescent microscopic images of the gel film surface before light irradiation are overlaid; (b) A similar overlaid image after the surface was washed after light irradiation. The scale bar is 100 μm. DETAILED DESCRIPTION OF THE INVENTION

[0018] The following describes embodiments of the present invention. The scope of the present invention is not limited to these descriptions, and other than the following examples, the present invention can be implemented with appropriate modifications within the scope that does not depart from the spirit of the present invention.

[0019] 1. Materials for immobilizing biological materials The biological material of the present invention is a material for immobilizing a biological material having a lipid membrane on a substrate, and as shown in FIG. (A) a hydrogel thin film layer made of a polymer; and (B) Biomaterial-immobilized compounds modified on the hydrogel thin film layer The biological material immobilizing compound (B) is characterized by having: (a) a hydrophobic chain having the function of interacting with and binding to a biological substance; and (b) Hydrophilic chains connected to the surface of the hydrogel thin film layer Here, the reactive group X at the end of the hydrophilic chain (b) is covalently bonded to the reactive group Y at the end of the polymer that makes up the hydrogel thin film layer (A), thereby linking the biosubstance immobilization compound (B) to the surface of the hydrogel thin film layer (A). As shown in Figure 1 (right), the hydrophobic chain (a) can bind to and capture biosubstances through interactions such as hydrophobic interactions, thereby immobilizing the biosubstances in specific regions on the surface of the immobilization material.

[0020] The biological material to be immobilized in the present invention can be a wide range of structures having a lipid membrane. The lipid membrane interacts with the hydrophobic chain of the biological material immobilization compound (B), allowing the biological material to be immobilized on the surface of the material. Here, "lipid membrane" refers to a membrane-like lipid. In this specification, "lipid" refers to a group of substances that are poorly soluble in water but readily soluble in organic solvents. While lipids typically include long-chain fatty acids and their derivatives or analogs, this specification also encompasses organic compounds such as steroids, carotenoids, terpenoids, isoprenoids, and fat-soluble vitamins. Examples of lipids include, but are not limited to, simple lipids (esters of fatty acids and alcohols, also known as neutral lipids; examples include fats and oils (triacylglycerols), waxes (fatty acid esters of higher alcohols), sterol esters, and fatty acid esters of vitamins); complex lipids (compounds that have an ester bond or an amide bond and, in addition to fatty acids and alcohols, have polar groups such as phosphate, sugar, sulfuric acid, and amine; examples include phospholipids (glycerophospholipids and sphingophospholipids), glycolipids (glyceroglycolipids and sphingoglycolipids), lipoproteins, and sulfolipids); and derived lipids (fat-soluble compounds produced by hydrolysis of simple lipids and complex lipids; examples include fatty acids, higher alcohols, fat-soluble vitamins, steroids, and hydrocarbons).

[0021] Examples of biological materials having such lipid membranes include cells, organelles, vesicles, viruses, liposomes, and micelles. Here, "cells" can include animal cells, plant cells, insect cells, prokaryotic cells, fungal cells, and the like. These include "suspension cells" (e.g., blood cells) that grow in a suspended or sedimented state without adhering or spreading on the surface of a support such as a culture vessel, "adherent cells" (e.g., fibroblasts detached from a support with an EDTA solution) that are dispersed from the support and temporarily suspended, and the like, adhered to the support. Also included are liposomes, exosomes, bacteria, viruses, organelles, and plant cells (protoplasts) with their cell walls removed, which have phospholipid bilayers on their surfaces. In addition, the immobilization material of the present invention can also immobilize lipid-containing substances, such as lipid-coated particles.

[0022] 1-1. Biological material immobilization compound (B) First, among the components constituting the biological substance immobilization material of the present invention, the biological substance immobilization compound (B) will be described. As shown in Figure 2(1), the biological substance immobilization compound (B) has a structure in which a hydrophobic chain (a) and a hydrophilic chain (b) are linked together, and these may be linked by a linker part (c).

[0023] The hydrophobic chain (a) of the biological substance immobilization compound is a site that binds to a target biological substance through interaction and captures the biological substance. Such interaction can be a non-covalent interaction such as a hydrophobic interaction. Specifically, the hydrophobic chain (a) can bind to a target cell through a hydrophobic interaction with the lipid portion of a lipid bilayer membrane such as a cell membrane.

[0024] The hydrophobic chain (a) is not particularly limited as long as it can bind to a biological substance through hydrophobic interaction, but it can be a saturated or unsaturated hydrocarbon chain which may have a substituent. Examples of such hydrocarbon chains include, for example, C 7-30 Alkyl group (preferably C 7-22alkyl group), C 6-14 Aryl group, C 6-14 Aryl C 7-30 Alkyl group (preferably C 6-14 Aryl C 7-22 alkyl group), and C 7-30 Alkyl C 6-14 Aryl groups (preferably C 6-14 Aryl C 7-22 Preferably, the alkyl group is a C group in which adjacent carbon atoms may be connected by 1 to 3 unsaturated bonds. 7-30 Alkyl group, C in which adjacent carbon atoms may be connected by 1 to 3 unsaturated bonds 7-22 an alkyl group, or a C group in which adjacent carbon atoms may be connected by 1 to 3 unsaturated bonds; 11-22 an alkyl group, or a C group in which adjacent carbon atoms may be connected by 1 to 3 unsaturated bonds; 16-18 It can be an alkyl group. More preferably, the hydrophobic chain (a) can be 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, a cis-9-octadecenyl (oleyl) group, a cis,cis-9,12-octadecadienyl (linolenyl) group, or a (9E,12E,15E)-octadeca-9,12,15-trienyl (elaidolinolenyl) group. In particular, an oleyl group, which is a part of phospholipids constituting cell membranes, is preferred. Furthermore, these hydrophobic chains may be substituted with any substituent and may contain heteroatoms such as N, S, and O.

[0025] In the present specification, when it is defined as "optionally having a substituent," the type, substitution position, and number of the substituent are not particularly limited, and when two or more substituents are present, 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, and an oxo group. These substituents may further have a substituent.

[0026] 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, as well as polymers and copolymers of glycolic acid derivatives, lactic acid derivatives, and p-dioxane derivatives. The polyalkylene glycol is preferably a polymer of oxyalkylene units having 2 to 4 carbon atoms, and the average polymerization number is in the range of 2 to 500 (preferably 45 to 500). The hydrophilic polymer is preferably a biocompatible polymer, more preferably polyethylene glycol (PEG). The polyethylene glycol preferably has an average molecular weight of 2,000 or more. The hydrophilic chain (b) may further have any substituent.

[0027] For the purpose of linking the biological substance-immobilizing compound (B) to the surface of the hydrogel thin film layer (A), as described above, the hydrophilic chain (b) has, within its molecule, preferably at its end, a reactive group X capable of covalently bonding with a reactive group Y at the end of the polymer constituting the biological substance hydrogel thin film layer. The reactive group X is preferably a functional group capable of covalently bonding with the reactive group Y, and any known functional group can be used in relation to the reactive group Y that can be introduced into the polymer described below. However, it is preferred that the reactive group X and the reactive group Y are a combination capable of forming a covalent bond in a hydrophilic solvent environment such as water.

[0028] For example, examples of combinations of reactive groups X and Y include an amino group and an N-hydroxysuccinimidyl (NHS) group, an amino group and a sulfosuccinimidyl group, an amino group and a nitrophenyl ester group, an azide group and an alkyne group (cycloaddition reaction), an azide group and a dibenzocyclooctyne group (cycloaddition reaction), a thiol group and a maleimide group (Michael addition reaction), a thiol group and an iodoacetamide group, a thiol group and a vinyl sulfone group, an aldehyde group and a hydrazine group, a ketone group and a hydrazine group, an aldehyde group and an aminooxy group, and a ketone group and an aminooxy group. These combinations can be used by interchangeably replacing the reactive groups X and Y.

[0029] In a preferred embodiment, a combination in which the reactive group X is a nucleophilic functional group and the reactive group Y is an electrophilic functional group; or a combination in which the reactive group X is an electrophilic functional group and the reactive group Y is a nucleophilic functional group can be used.

[0030] Examples of such nucleophilic functional groups include thiol groups (-SH) and amino groups, and those skilled in the art can appropriately use known nucleophilic functional groups. Furthermore, active ester groups can be used as electrophilic functional groups. Examples of electrophilic functional groups include maleimidyl groups, N-hydroxysuccinimidyl (NHS) groups, sulfosuccinimidyl groups, phthalimidyl groups, imidazoyl groups, acryloyl groups, -CO2PhNO2 (where Ph represents o-, m-, or p-phenylene), and nitrophenyl groups, and those skilled in the art can appropriately use other known active ester groups.

[0031] 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. Conversely, 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 the amino group (reactive group Y) in collagen, and particularly preferably has an NHS group.

[0032] The linker portion (c) connecting the hydrophobic chain (a) and the hydrophilic chain (b) can be a functional group capable of forming 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. Linker structures such as oligomers or polymers having such functional groups can also be used, or, as described below, a linker having a branched chain can also be used. Examples of branched linkers (c) include trihydric alcohols such as glycerol; benzenetriols or benzenetricarboxylic acids such as hydroxyquinol; benzenetriamine; and 4-aminosalicylic acid, each of which has a benzene ring with three or more reactive functional groups.

[0033] In a preferred embodiment, as shown in Figure 2(2), the biological substance immobilization compound (B) can further have, in a side chain branching from the linker portion, (d) a binding-inhibiting group that inhibits binding between the biological substance and the hydrophobic chain; and (e) a photoreactive group whose bond is cleaved or whose structure is changed by a photoreaction. Typically, in such an embodiment, by irradiating the biological substance immobilization compound (B) with light such as visible light or ultraviolet light, the photoreactive group is detached from the linker portion while remaining linked to the binding-inhibiting group; thereby, the binding inhibition by the binding-inhibiting group is eliminated, and the hydrophobic chain becomes capable of binding to the biological substance. In other words, a switching function can be imparted, allowing the biological substance to be immobilized and positioned only in the desired region irradiated with light.

[0034] In this specification, the term "photoreactive group" refers to a group in which the bond in the photoreactive group is cleaved or the structure thereof is changed by irradiation with light such as visible light or ultraviolet light. Such a photoreactive group is not particularly limited as long as it can eliminate the binding inhibition caused by a binding-inhibiting group by photoreaction, and for example, a divalent group having a 2-nitrobenzyl skeleton, a nitrophenyl ethyl ester skeleton, a coumarin-4-ylmethyl skeleton, a phenylcarbonylmethyl skeleton, or a 7-nitroindolinocarbonyl skeleton can be used. A divalent group having a 2-nitrobenzyl skeleton is preferred.

[0035] In this specification, the term "divalent group having a 2-nitrobenzyl skeleton" refers to a divalent group having the following structure or a derivative structure thereof. [ka] [wherein the arrow on the right indicates a link to the linker portion, the arrow on the left indicates a link to the binding inhibitor group, and L 6 is an ethynylene group or is absent.

[0036] As the divalent group having a 2-nitrobenzyl skeleton, the following are preferred. [ka] [In the formula, the arrow on the right indicates a link to a linker moiety, and the arrow on the left indicates a link to a binding-inhibiting group.]

[0037] In this specification, the term "divalent group having a coumarin-4-ylmethyl skeleton" refers to a divalent group having the following structure or a derivative structure thereof: [ka] [wherein the arrow on the right indicates a link to the linker portion, the arrow on the left indicates a link to the binding inhibitor group, and L 7 is C 1-10an alkylene group or no alkylene group, wherein carbon atoms in the alkylene group may be substituted with 1 to 5 oxo groups, adjacent carbon atoms may be connected to each other by 1 to 5 unsaturated bonds, and 1 to 4 of the carbon atoms in the alkylene group are substituted with NH, N(C 1-10 alkyl), O or S.

[0038] As the divalent group having a coumarin-4-ylmethyl skeleton, the following are preferred. [ka] [In the formula, the arrow on the right indicates a link to a linker moiety, and the arrow on the left indicates a link to a binding-inhibiting group.]

[0039] In this specification, the term "divalent group having a phenylcarbonylmethyl skeleton" refers to a divalent group having the following structure or a derivative structure thereof: [ka] [In the formula, the arrow on the right indicates a link to a linker moiety, and the arrow on the left indicates a link to a binding-inhibiting group.]

[0040] As the divalent group having a phenylcarbonylmethyl skeleton, the following are preferred. [ka] [In the formula, the arrow on the right indicates a link to a linker moiety, and the arrow on the left indicates a link to a binding-inhibiting group.]

[0041] In this specification, the term "divalent group having a 7-nitroindolinocarbonyl skeleton" refers to a divalent group having the following structure or a derivative structure thereof. [ka] [In the formula, the arrow on the right indicates a link to a linker moiety, and the arrow on the left indicates a link to a binding-inhibiting group.]

[0042] As the divalent group having a 7-nitroindolinocarbonyl skeleton, the following are preferred. [ka] [In the formula, the arrow on the right indicates a link to a linker moiety, and the arrow on the left indicates a link to a binding-inhibiting group.]

[0043] In another embodiment, a mechanism may be used in which a photoreaction changes the structure of the photoreactive group, thereby eliminating the binding inhibition caused by the binding inhibiting group. In this case, the photoreactive group is not particularly limited as long as it eliminates the binding inhibition caused by the binding inhibiting group by a photoreaction, and for example, a divalent group having an azobenzene skeleton, a fulgide skeleton, a spiropyran skeleton, a spirooxazine skeleton, or a diarylethene skeleton can be used.

[0044] Other specific examples of photoreactive groups include dimethoxynitrobenzyl ester group (DMNB), bromohydroxycoumarin (Bhc) group, dimethoxybenzoin group, 2-nitropiperonyloxycarbonyl (NPOC) group, 2-nitroveratryloxycarbonyl (NVOC) group, α-methyl-2-nitropiperonyloxycarbonyl (MeNPOC) group, α-methyl-2-nitroveratryloxycarbonyl (MeNVOC) group, 2,6-dinitrobenzyloxycarbonyl (DNBOC) group, α-methyl-2,6-dinitrobenzyloxycarbonyl (MeDNBOC) group, 1-(2-nitrophenyl)ethyloxycarbonyl (NPEOC) group, 1-methyl- ... Examples of the alkyl group include a 3'-anthracenyl)ethyloxycarbonyl (MeNPEOC) group, a 9-anthracenylmethyloxycarbonyl (ANMOC) group, a 1-pyrenylmethyloxycarbonyl (PYMOC) group, a 3'-methoxybenzoinyloxycarbonyl (MBOC) group, a 3',5'-dimethoxybenzoyloxycarbonyl (DMBOC) group, a 7-nitroindolinyloxycarbonyl (NIOC) group, a 5,7-dinitroindolinyloxycarbonyl (DNIOC) group, a 2-anthraquinonylmethyloxycarbonyl (AQMOC) group, an α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl group, and a 5-bromo-7-nitroindolinyloxycarbonyl (BNIOC) group.

[0045] In addition, in this specification, the term "binding inhibitor group" refers to a group that physically or chemically inhibits the binding of the hydrophobic chain (a) to a lipid membrane. The structure of the binding inhibitor group may be the same as or different from the structure of the hydrophobic chain (a). The binding inhibitor group is not particularly limited, but may be a saturated or unsaturated hydrocarbon chain that may have a substituent. Examples of such hydrocarbon chains are as described above for the hydrophobic chain (a). The binding inhibitor group (d) and the photoreactive group (e) may be linked directly, or they may be linked via any spacer group.

[0046] Specific examples of immobilized compounds having such a binding-inhibiting group (d) and a photoreactive group (e) include the compounds disclosed in WO2016 / 158327.

[0047] 1-2. Hydrogel thin film layer (A) Next, the hydrogel thin film layer (A), one of the components constituting the biological material of the present invention, will be described. The hydrogel thin film layer (A) can typically be produced by forming a thin film of hydrogel on the surface of a substrate, as described below.

[0048] The hydrogel thin film layer (A) can be a hydrogel formed by gelling polymers such as hydrophilic polymers through intermolecular cross-linking. In this specification, the term "gel" broadly refers to a dispersion system of polymers that has high viscosity and has lost fluidity, and the term "hydrogel" refers to a gel containing water as a solvent (dispersion medium). Hydrogels with a network structure, particularly a three-dimensional network structure, are preferred.

[0049] The polymer used to form 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 polymers used in the hydrogel thin film layer (A) include carbohydrate polymers (methylcellulose, carboxymethylcellulose, hydroxymethylcellulose, hydroxypropylcellulose, hydroxyethylcellulose, ethylcellulose, dextrin, cyclodextrin, alginate, hyaluronic acid, chitosan, etc.), protein polymers (gelatin, albumin, collagen, glycol protein, etc.), hydroxy acid polyesters (biodegradable polylactide-co-glycolide (PLGA), polylactic acid (PLA), polyglycolide, polyhydroxybutyric acid, polycaprolactone, polyvalerolactone, polyphosphazene, polyorthoester, etc.), polyanhydrides, polyethylene glycol, polyvinyl polyhydroxyalkyl methacrylate, polyvinylpyrrolidone, polyvinyl alcohol, and polyamides. Preferably, polyalkylene glycol, cellulose, a cellulose derivative, hyaluronic acid, chitosan, or collagen can be used.

[0050] Among these, polyethylene glycol (PEG) is preferred as a biocompatible polymer, and 2-, 4-, or 8-branched polyethylene glycol (PEG) is particularly preferred. Such PEG has little effect on cells, and is therefore advantageous in that it allows cells to be recovered without impairing their inherent functions (i.e., cells can be recovered while still 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.

[0051] It is known in the art that hydrogels can be obtained by crosslinking polyethylene glycols having branched structures such as di-, tetra-, or octa-branched structures. More specifically, a preferred hydrogel is obtained by mixing a first polymer unit (PEG) having one or more nucleophilic functional groups at its side chains or terminals with a second polymer unit (PEG) having one or more electrophilic functional groups at its side chains or terminals in a solution, and then crosslinking these polymer units. In this case, unreacted nucleophilic or electrophilic functional groups from the polymer units remain in the hydrogel even after the hydrogel is formed, and these remain as reactive groups Y to bond with reactive groups X in the hydrophilic chain (b).

[0052] A particularly preferred polymer unit is tetra-branched polyethylene glycol (Tetra-PEG). Gels consisting of such a tetra-branched polyethylene glycol backbone are commonly known as Tetra-PEG gels. A network structure is constructed by an AB cross-end coupling reaction between two types of tetra-branched polymers, each of which has an electrophilic functional group, such as an activated ester structure, and a nucleophilic functional group, such as an amino group, at its terminal (Matsunaga et al., Macromolecules, Vol. 42, No. 4, pp. 1344-1351, 2009). Tetra-PEG gels can be easily prepared in situ by simply mixing the two polymer solutions, and the gelation time can be controlled by adjusting the pH and ionic strength during gel preparation.

[0053] Here, the types of nucleophilic functional groups and electrophilic functional groups 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. Alternatively, 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.

[0054] In addition to the combination of nucleophilic and electrophilic functional groups, combinations of reactive groups capable of forming chemical bonds between polymer units in solution can also be used, such as combinations of azide and alkyne groups (cycloaddition reaction), azide and dibenzocyclooctyne groups (cycloaddition reaction), thiol and iodoacetamide groups, thiol and vinyl sulfone groups, aldehyde and hydrazine groups, ketone and hydrazine groups, aldehyde and aminooxy groups, and ketone and aminooxy groups.

[0055] In a preferred embodiment, the hydrogel constituting the hydrogel thin film layer (A) can be a hydrogel formed from a polymer having photodegradable groups in its molecule (photodegradable hydrogel). In this case, the photodegradable groups are cleaved by irradiation with light, causing the hydrogel thin film layer to decompose. Therefore, after immobilizing the target biological material, the desired region can be irradiated with visible light, ultraviolet light, or the like, to selectively recover only the cells immobilized in that region.

[0056] As such a photodegradable group, a divalent group having a skeleton selected from the group consisting of a 2-nitrobenzyl skeleton, a nitrophenylethyl ester skeleton, a coumarin-4-ylmethyl skeleton, a phenylcarbonylmethyl skeleton, a 7-nitroindolinocarbonyl skeleton, an azobenzene skeleton, a fulgide skeleton, a spiropyran skeleton, a spirooxazine skeleton, and a diarylethene skeleton can be used. Other specific examples of the photodegradable group include a dimethoxynitrobenzyl ester group (DMNB), a bromohydroxycoumarin (Bhc) group, a dimethoxybenzoin group, a 2-nitropiperonyloxycarbonyl (NPOC) group, a 2-nitroveratryloxycarbonyl (NVOC) group, an α-methyl-2-nitropiperonyloxycarbonyl (MeNPOC) group, an α-methyl-2-nitroveratryloxycarbonyl (MeNVOC) group, a 2,6-dinitrobenzyloxycarbonyl (DNBOC) group, an α-methyl-2,6-dinitrobenzyloxycarbonyl (MeDNBOC) group, a 1-(2-nitrophenyl)ethyloxycarbonyl (NPEOC) group, a 1-methyl- ... Examples of the alkyl group include a 3'-phenylethyloxycarbonyl (MeNPEOC) group, a 9-anthracenylmethyloxycarbonyl (ANMOC) group, a 1-pyrenylmethyloxycarbonyl (PYMOC) group, a 3'-methoxybenzoinyloxycarbonyl (MBOC) group, a 3',5'-dimethoxybenzoyloxycarbonyl (DMBOC) group, a 7-nitroindolinyloxycarbonyl (NIOC) group, a 5,7-dinitroindolinyloxycarbonyl (DNIOC) group, a 2-anthraquinonylmethyloxycarbonyl (AQMOC) group, an α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl group, and a 5-bromo-7-nitroindolinyloxycarbonyl (BNIOC) group.

[0057] By introducing these photodegradable groups into a polymer, a photodegradable hydrogel can be obtained. Such an introduction method can be achieved by chemical synthesis known in the art. For example, when using the above-mentioned Tetra-PEG gel, a photodegradable hydrogel can be obtained by introducing a photodegradable group into one or more branched chains in the Tetra-PEG polymer unit. A specific example is Tetra-PEG in which the four-branched branched chain has the following structure: [ka]

[0058] 2. Substrate for immobilizing biological substances and manufacturing method The present invention also relates to a substrate for immobilizing a biological substance, the surface of which has been modified with the above-mentioned immobilization material. The immobilization substrate has a structure in which a hydrogel thin film layer (B) and a biological substance immobilization compound (A) are present in this order on the substrate, as shown in Figure 1.

[0059] The material and shape of the substrate to be modified with the substrate for immobilizing a biological material of the present invention are not particularly limited, and various appropriate substrates can be selected depending on the intended use. For example, the shape of the substrate to be modified may be a substrate (plate-shaped or film-shaped, such as a glass slide, dish, microplate, or microarray substrate), a carrier (particulate or colloidal, such as beads), a fibrous structure, a tube, or a container (e.g., a test tube or vial). Examples of materials for the substrate to be modified include glass; cement; ceramics or fine ceramics such as porcelain; polymer resins such as polyethylene terephthalate, cellulose acetate, polycarbonate, polystyrene, and polymethyl methacrylate; biological materials such as polypeptides and proteins; silicon; activated carbon; porous glass; porous ceramics; porous silicon; porous activated carbon; nonwoven fabrics; filter paper; membrane filters; and conductive materials such as gold. The surface of the substrate to be modified may be coated with a polymer such as a polycation in order to introduce an amino group, a carboxyl group, a hydroxyl group, or the like, or may be treated with a silane coupling agent having a substituent group to be introduced into the substrate surface, or reactive functional groups may be introduced by plasma treatment.

[0060] A substrate for immobilizing a biological material having a surface modified with the immobilization material of the present invention can typically be produced by the process shown in Figure 3(i). That is, the method for producing a substrate for immobilizing a biological material comprises the following steps: (p) modifying the entire surface of the substrate with a thin film layer of a hydrogel made of a polymer; and (q) modifying the surface of the hydrogel thin film layer with a biological substance immobilizing compound 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.

[0061] The surface modification in step (p) can be carried out, for example, by applying a solution containing a polymer or hydrogel to the surface of the substrate. Such application can be performed using techniques known in the art, such as a spacer or a bar coder. The solution to be applied can contain optional additives, such as a crosslinking agent for gelling the polymer and a pH adjuster.

[0062] Step (q) can be carried out by dropping or applying a solution containing an immobilized compound having a hydrophobic chain and a hydrophilic chain onto the surface of the hydrogel thin film layer formed in step (p). As described above, the reactive group X of the hydrophilic chain forms a covalent bond with the reactive group Y of the hydrogel thin film layer, thereby modifying the surface of the hydrogel thin film layer with the immobilized compound. The concentration of the immobilized compound in the solution can be in the range of 3 to 2000 μM.

[0063] In a preferred embodiment, in the method for producing a substrate for immobilizing a biological material, a photoresponsive immobilization compound is used to provide a patterned surface modification that exhibits binding to a biological material only in areas irradiated with light such as visible light or ultraviolet light, thereby enabling the immobilization of a target biological material in a specific area on the substrate surface. For example, multiple spot-shaped modification areas can be provided to immobilize a single cell. The diameter of the spot for immobilizing a single cell can be approximately 2 to 30 μm or 5 to 15 μm, depending on the size of the target cell. Here, the term "photoresponsive immobilization compound" refers to an immobilization compound having a binding inhibitor group that inhibits binding between a biological material and a hydrophobic chain, and a photoreactive group whose bond is cleaved or whose structure is changed by a photoreaction, as described above (the binding inhibitor group and the photoreactive group are as described above).

[0064] Such a substrate for immobilizing a biological material having a patterned surface modification can be prepared by further carrying out the following step (r) after the above step (q), as shown in FIG. 3(ii): (r) A step of patterning the surface of a substrate so that only specific regions on the substrate are endowed with the ability to bind to biological substances, the step comprising irradiating the specific regions on the substrate surface with light to eliminate binding inhibition by the binding-inhibiting groups.

[0065] In step (r), the wavelength of the light to be irradiated can be determined depending on the type of photoreactive group. Light with a wavelength in the range of 157 to 600 nm, preferably around 250 to 450 nm, is typically used. However, when photoreaction is carried out by multiphoton absorption, light with a longer wavelength than the above can also be used. Light sources that can be used include sunlight, electric light such as mercury lamps, laser light (semiconductor lasers, solid-state lasers, and gas lasers), light emitted by light-emitting diodes, and light emitted by electroluminescent devices. Light irradiation can be performed by uniformly irradiating the substrate surface with light from a light source through an appropriate filter as needed, or by pattern exposure of the desired shape using a so-called mask. Alternatively, light can be focused using a lens or mirror and irradiated in a fine pattern. Alternatively, scanning exposure with the focused light beam can be performed. Pattern exposure can also be performed by contact exposure, which is an exposure method in which a mask (reticle) and a workpiece (sample) are brought into contact with each other. Alternatively, proximity exposure, a non-contact exposure method in which exposure is performed by setting the gap between the mask (reticle) and the workpiece (sample) at a few μm to several tens of μm, may be used. Furthermore, a projection exposure method (maskless exposure method) in which an image created using a liquid crystal or digital mirror device is projected onto the workpiece surface may also 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 biological materials such as cells is present on the workpiece. The light irradiation energy can be set as appropriate, but is usually 0.001 to 1000 J / cm. 2 and 0.01 to 100 J / cm 2 is preferred.

[0066] 3. Immobilization and recovery methods for biological materials Furthermore, the present invention also relates to a cell sorting technique in which target cells are immobilized and selectively recovered using a cell immobilization substrate whose surface has been modified with a photodegradable cell immobilization agent. The cell recovery method of the present invention comprises the following steps: (m) contacting a solution containing a predetermined target biological substance with a biological substance immobilization substrate to immobilize the target biological substance on the immobilization substrate; (n) A step of separating and recovering the immobilized target biological substance from the immobilization substrate.

[0067] The separation of the biological material in step (n) can be carried out by washing the substrate, and the washing conditions and the like can be determined by techniques known in the art.

[0068] The method can be carried out by placing a cell immobilization substrate in a microchannel. In this case, in step (n), a flux is applied to the substrate surface, and the target cells separated from the cell immobilization substrate can be collected.

[0069] When the surface of a substrate is patterned using the above-mentioned photoresponsive immobilization compound, it is possible to immobilize and recover a single cell by creating multiple spot-type modified regions for immobilizing a single cell.

[0070] Alternatively, a hydrogel thin film layer (photodissolvable gel thin film) formed from a polymer having photodegradable groups in its molecule can be used. In this case, the hydrogel thin film layer is degraded by cleaving the photodegradable groups upon irradiation with light. Therefore, after immobilizing the target biological material, the desired area can be irradiated with visible light or ultraviolet light, allowing for the location-selective recovery of only the cells immobilized at that location (Figure 3(iii)). ).

[0071] 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 above step (n): (n') A step of irradiating a specific region of the immobilization substrate with light to decompose the hydrogel thin film layer, thereby separating and recovering only the target biological substance immobilized in the specific region from the immobilization substrate.

[0072] In this case, the light irradiation conditions may be determined depending on the type of photodegradable group, and can be the same as those in step (r) above. Furthermore, the separation of the biological material in step (n') can be carried out by washing the substrate, and the solution conditions for such washing can be determined by methods known in the art. [Example]

[0073] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0074] 1. Hydrogel Thin Film Preparation Hydrogels were prepared by mixing a 4-branched PEG (molecular weight approximately 10,000, NOF Corp., Sunbright PTE100GS) bearing N-hydroxysuccinimide (NHS) ester at its termini and a 4-branched PEG (molecular weight approximately 10,000, NOF Corp., Sunbright PTE100PA) bearing amino groups at their termini in aqueous solution to a final concentration of 5 mM. Hydrogels were also prepared using gelatin (Sigma) at a final concentration of 5 wt% instead of the 4-branched PEG bearing amino groups. To prepare a flat gel film on a glass substrate, two 25 μm-thick silicone strips (approximately 5 mm × 20 mm) were attached parallel to the glass substrate, spaced approximately 10 mm apart. A glass slide was then attached to the silicone strips, creating a 25 μm-high cavity. The hydrogel mixture was then rapidly poured into the cavity and allowed to stand for at least 15 minutes. A small amount of fluorescent beads with a diameter of approximately 1 μm was suspended in this mixture in advance, and gelation was confirmed by observing the Brownian motion of the fluorescent beads under a fluorescence microscope (gel formation was confirmed when the Brownian motion stopped).

[0075] To prepare a thin, uniform gel film over the entire substrate surface, we also used a bar coater. After allowing the mixture of gel materials to react for 9 minutes at 30°C (if the reaction lasted longer than 10 minutes, the solution would gel before spreading; on the other hand, if the reaction time was too short, the viscosity would be insufficient to spread the film over a flat surface), the mixture was dropped onto the edge of a glass substrate and spread using a bar coater (manufactured by AS ONE Corporation, for 1.5 μm thin films). The mixture was then left to stand for approximately 20 minutes at room temperature in a saturated water vapor environment.

[0076] 2. Modification of immobilization materials onto thin gel films and immobilization of cells The hydrogel thin film was modified with PEG lipid (NOF Corporation, Sunbright OE-040CS) having the following structure as an immobilization compound by reacting it with the amino groups on the hydrogel thin film prepared in step 1. The PEG lipid used is a compound with a lipid chain as the hydrophobic chain and a polyethylene glycol (PEG) chain as the hydrophilic chain, and an NHS ester group at the end of the hydrophilic chain that can react with the NH group on the surface of the gel thin film. [ka]

[0077] After preparing the gel thin film using the silicon spacer described above, the slide glass was removed, and 5 μl of 20–500 μM PEG-lipid aqueous solution was immediately added dropwise to the exposed surface. The reaction was allowed to proceed at 37°C for 3 hours under saturated water vapor. The gel thin film surface was then washed with phosphate buffer solution (PBS) to remove unreacted PEG-lipid. A suspension of mouse proB cells, the BaF3 strain, expressing green fluorescent protein (EGFP) (EGFP-BaF3 cells), was added dropwise to the gel thin film surface and allowed to stand for 15 minutes before being removed. After further washing with PBS to remove nonspecifically adsorbed cells, the gel thin film surface was observed under a fluorescence microscope. Cells were immobilized only on the areas modified with PEG-lipid (Figure 4a, b). Furthermore, when the observation focus was gradually raised from the glass surface under higher magnification, an image was observed in which the fluorescent beads attached to the glass were in focus at the glass surface (Fig. 4c), an image in which the fluorescent beads dispersed in the gel were in focus 15 μm above the glass surface (Fig. 4d), and an image in which the bottom of the EGFP-BaF3 cells were in focus 25 μm above the glass surface (Fig. 4e). This confirmed that the cells were fixed onto the approximately 25 μm thick gel film via the PEG lipid.

[0078] We also observed thin gel films with PEG-lipid modified concentrations of 50, 100, and 500 μM. Similarly, we observed cell fixation only in the PEG-lipid modified area, and there was no significant difference in the density of the fixed cells (Figure 5a-c).

[0079] On the other hand, after preparing the gel thin film, the slide glass was removed and left in a clean bench for over 30 minutes to dry. We then attempted similar PEG-lipid modification and cell fixation. However, very few cells were fixed. Observation of the dried gel surface revealed that the polymers of the gel material formed fibrous aggregates several tens of micrometers thick. Furthermore, even after re-immersion in PBS to swell, the thick fibrous aggregates did not dissolve. These results suggest that if the gel material aggregates upon drying after preparation, the PEG-lipid cannot be sufficiently modified on the gel thin film surface, preventing cell fixation. This suggests that when using a gel thin film approximately 25 μm thick, a gel thin film with a high cell density can be prepared by modifying the gel with a PEG-lipid concentration of 20–500 μM before drying.

[0080] We also performed a similar experiment on a hydrogel thin film made of a 4-arm PEG with an NHS ester and gelatin. Similarly, cell fixation was observed only in the PEG-lipid-modified region (Figure 6a, b). As described above, by gradually raising the focus from the glass surface, we observed an image of the gelatin gel where the focus was on the bottom of the EGFP-BaF3 cells 15 μm above the glass surface (Figure 6c) (Figure 6d). This indicates that when gelatin is used as the material, the gel shrinks slightly, but this does not affect cell fixation, demonstrating that this method can fix cells on a thin gel film. Thus, despite the differences in hydrogel materials and properties, this method was able to fix cells at high densities on a thin hydrogel film.

[0081] 3. Immobilization of cells on a thin gel film in a microchannel Observation and analysis of cells in microchannels is inexpensive and reproducible, reducing the consumption of expensive media and chemicals, enabling uniform cleaning of the cell culture surface, and simplifying and automating the process. Therefore, we created a channel by covering a commercially available microchamber (ibidi Sticky-Slide VI 0.4) with a thin gel film prepared on a glass slide using a bar coater as described above. The gel was prepared by mixing the two four-arm PEG materials (final concentration 5 mM). A small amount of fluorescein-5-NHS ester (final concentration 50 μM) was also added to enable fluorescence microscopy. A 10–100 μM aqueous solution of PEG-lipid was injected into the microchannel and reacted at 37°C for 3 hours. To remove unreacted PEG-lipid, the gel surface was washed with PBS. To block the remaining amino groups on the gel surface, a 5 mM sulfo-NHS acetate solution was injected into the channel and allowed to react for 3 hours at 37°C. The gel surface was then washed again with PBS. A suspension of BaF3 cells expressing a red fluorescent protein (Kusabira Orange: KO) (KO-BaF3 cells) was then injected into the channel and allowed to stand for 10 minutes before being removed. PBS was then injected five times to wash the gel surface. Nonspecifically adsorbed cells were then removed, and the gel surface was then observed using a confocal laser microscope.

[0082] Bright-field and red fluorescent images confirmed that cells were densely and uniformly immobilized throughout the entire channel, exclusively on the gel surface of the PEG-lipid-modified channel (Figure 7a-c). Furthermore, when observing the 10 μM PEG-lipid-modified channel at higher magnification and by raising the observation focus approximately 2 μm above the glass surface, a thin gel film with green fluorescence approximately 4-8 μm thick was observed, followed by red-fluorescent cells approximately 8-12 μm thick (Figure 7d). These results demonstrate that cells can be rapidly and uniformly immobilized at high density on a thin gel film that is thin enough to prevent leakage of liquid from the gel layer, even when covered with a microchamber.

[0083] 4. Photo-positioning of cells on thin gel films (using photoactivatable PEG lipids) The surface of a thin gel film prepared in a microchannel by method 3 above was modified with photoactivatable PEG lipid (a compound of formula (Ic) described in WO2016 / 158327), and cells were placed only in the illuminated area of ​​the gel surface. A 100 μM aqueous solution of photoactivatable PEG lipid was injected into the microchannel on the thin gel film prepared in the same manner as above, and allowed to react at 37°C for 3 hours. This was then blocked with a 5 mM aqueous sulfo-NHS acetate solution in the same manner. This microchannel was placed on a photomask (line pattern with 400 μm spacing), and 360 nm light was applied from below using a xenon lamp at 1.5 J / cm. 2 The KO-BaF3 cell suspension was then injected into the channel and immobilized on the gel surface in the same manner as above. PBS was injected five times to wash the surface of the thin gel film, and nonspecifically adsorbed cells were removed. The surface of the thin gel film was then observed using a confocal laser microscope. As a result, a line pattern of cells was observed on the gel surface, and it was confirmed that cells were arranged only in the light-irradiated area (Figure 8a, b). Furthermore, when the boundary between the light-irradiated and non-irradiated areas was photographed at high magnification by shifting the focus from the bottom of the channel upward, it was confirmed that red cells were arranged on the green thin gel film depending on whether or not light was irradiated (Figure 8c). This result demonstrates that the present invention can arrange cells in desired positions on the thin gel film.

[0084] 5. Cell detachment from a photodegradable gel film (example using a photodegradable hydrogel film) A photodissolvable thin gel film was fabricated in a microchannel using the same method as in 3 above. In this case, instead of the 4-branched PEG with NHS ester at the end, a 4-branched PEG with NHS ester at the end via a photodegradable linker (Figure 9a) was used, and the 4-branched PEG with amino groups at the end was mixed with a small amount of fluorescein-5-NHS ester. PEG lipids were modified on this photodissolvable thin gel film using the same method as above, and after blocking with sulfo-NHS acetate, KO-BaF3 cells were immobilized. Subsequently, the cells immobilized on the gel surface were irradiated with line-pattern light (5 J / cm) using a photomask in the same way as above. 2) and washed the gel surface with PBS to selectively recover only the cells in the light-irradiated area from the bottom of the channel.

[0085] As a result, the cells, which had been uniformly fixed over the entire gel surface before light irradiation, were removed from the irradiated area as the gel dissolved upon light irradiation, as confirmed by the patterns of green fluorescence from the gel and red fluorescence from the cells (Figure 9b, c). As in the above experiment, the irradiated and non-irradiated areas were enlarged and successive images were taken with the observation focus shifted upward. It was confirmed that cells remained fixed on the thin gel film in the non-irradiated area, but that the gel had disappeared in the irradiated area, along with almost all of the cells (Figure 9d). These results demonstrate that the present invention enables the rapid and simple immobilization of floating cells on a thin photodissolvable gel film, and that only cells in the desired positions among the immobilized cells can be isolated and recovered by light irradiation.

[0086] 6. Construction and culture of single cell arrays on thin gel films A thin gel film modified with photoactivatable PEG lipid was fabricated in a microchannel using the same method as in 4 above. This microchannel was placed on a photomask (a checkerboard pattern of circular light-transmitting regions with a diameter of 22 μm, spaced 100 μm apart). A xenon lamp was used to illuminate the microchannel from below with 360 nm light at 1.5 J / cm. 2 The KO-BaF3 cell suspension was then injected into the channel and immobilized on the gel surface in the same manner as above. PBS was injected five times to wash the surface of the thin gel film, and nonspecifically adsorbed cells were removed. The surface of the thin gel film was then observed using a confocal laser microscope. As a result, it was found that a single-cell array of KO-BaF3 cells could be constructed on the thin gel film, as shown in Figure 10.

[0087] Furthermore, a single-cell array was constructed using colon cancer B16-F10 cells, and cells were cultured on the array. Using the same method as in 4. above, a thin gel film was created in a microchannel using a 4-branched PEG with an NHS ester and gelatin, and modified by injecting 10 μM and 100 μM aqueous solutions of photoactivatable PEG lipids. Using the same method as above, a circular 360 nm light pattern (checkerboard pattern with 100 μm intervals) with a diameter of 24 μm was illuminated at 1.5 J / cm. 2 The cells were then irradiated with light. A B16-F10 cell suspension was then injected into the channel and immobilized on the gel surface in the same manner as above. The gel surface was washed five times with PBS to remove nonspecifically adsorbed cells, and the gel surface was then observed using a confocal laser scanning microscope. RPMI 1640 medium was then injected five times to replace the culture medium inside the channel. After culturing the cells at 37°C under a saturated water vapor environment with 5% carbon dioxide for 15 hours, the gel surface was again observed using a confocal laser scanning microscope. As shown in Figure 11, cell spreading was observed on the gel surface modified with 10 μM photoactivatable PEG-lipid solution after culturing. On the other hand, no cell spreading was observed on the gel surface modified with 100 μM. Thus, by modifying a gel thin film made with cell-adhesive gelatin with a low concentration of photoactivatable PEG-lipid, it was possible to culture single-cell arrays of adherent cells in an in vivo-like environment, and to analyze the adhesion and spreading state of adherent cells.

[0088] 7. Light harvesting at the single cell level using thin gel membranes Using the same method as in 5. above, a photolytic gel membrane modified with PEG-lipids was fabricated in a microchannel, and a mixed suspension of KO-BaF3 cells and EGFP-BaF3 cells was injected to immobilize two types of cells with different fluorescence characteristics. The surface of the gel membrane was observed using a confocal laser microscope, and a 405 nm laser beam, measuring the size of a single cell, was irradiated for 160 ms only at the locations where red-fluorescent KO-BaF3 cells were immobilized. The gel surface was then washed three times with PBS, allowing selective recovery of the irradiated KO-BaF3 cells from the gel surface. As shown in Figure 12, photoirradiation enabled selective recovery of KO-BaF3 cells at the single-cell level, demonstrating the feasibility of high-speed sorting.

Claims

1. An immobilization material for immobilizing a biological material having a lipid membrane on a substrate, comprising: The present invention has a hydrogel thin film layer made of a hydrogel having a structure in which polymer molecules are crosslinked with each other, and a structure modified with a biological material immobilization compound on the hydrogel thin film layer; the polymers are made of a first polymer having one or more nucleophilic functional groups on a side chain or at an end, and a second polymer having one or more electrophilic functional groups on a side chain or at an end; the first polymer is polyethylene glycol (PEG) having two branches, four branches, or eight branches, or a protein-based polymer; the second polymer is polyethylene glycol (PEG) having two branches, four branches, or eight branches, the biological material immobilization compound has a hydrophobic chain capable of interacting with the biological material and a hydrophilic chain connected to the surface of the hydrogel thin film layer; a reactive group X at the end of the hydrophilic chain, which can be covalently bonded to the surface of the hydrogel thin film layer; The polymer has a reactive group Y in the molecule that forms a covalent bond with the reactive group X. The immobilization material.

2. The immobilization material of claim 1 , wherein the hydrophilic chain comprises a polyalkylene glycol.

3. 3. The immobilization material according to claim 1, wherein the hydrophobic chain is a saturated or unsaturated hydrocarbon chain which may have a substituent.

4. The immobilization material according to any one of claims 1 to 3, wherein the combination of the reactive group X and the reactive group Y is selected from the group consisting of 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 azide group and an alkyne group; an azide group and a dibenzocyclooctyne group; a thiol group and a maleimide group; a thiol group and an iodoacetamide group; a thiol group and a vinyl sulfone group; an aldehyde group and a hydrazine group; a ketone group and a hydrazine group; an aldehyde group and an aminooxy group; or a ketone group and an aminooxy group.

5. 4. The immobilization material according to claim 1, wherein the reactive group X is a nucleophilic functional group and the reactive group Y is an electrophilic functional group.

6. 4. The immobilization material according to claim 1, wherein the reactive group X is an electrophilic functional group, and the reactive group Y is a nucleophilic functional group.

7. 7. The immobilization material according to claim 1, wherein the first and second polymers are both PEG having four branches (Tetra-PEG).

8. 7. The immobilization material according to claim 1, wherein the first polymer is gelatin, and the second polymer is PEG having four branches (Tetra-PEG).

9. In the first and second polymers, the nucleophilic functional groups are selected from the group consisting of thiol groups and amino groups; and the electrophilic functional groups are selected from the group consisting of maleimidyl groups, N-hydroxy-succinimidyl (NHS) groups, sulfosuccinimidyl groups, phthalimidyl groups, imidazoyl groups, acryloyl groups, -CO ２ PhNO ２ 9. The immobilization material according to claim 1, wherein the aryl group is selected from the group consisting of aryl, aryl ester ...

10. 10. The immobilization material according to claim 1, wherein in the first and second polymers, the nucleophilic functional groups are amino groups; and the electrophilic functional groups are N-hydroxy-succinimidyl (NHS) groups.

11. 11. The immobilization material of claim 10, wherein the reactive group X is an N-hydroxy-succinimidyl (NHS) group and the reactive group Y is an amino group.

12. 12. The immobilization material according to claim 1, wherein the biological substance immobilization compound has a linker portion connecting the hydrophobic chain and the hydrophilic chain.

13. the biological substance immobilization compound has, in a side chain branched from a linker portion, a bond-inhibiting group that inhibits the bond between the biological substance and the hydrophobic chain; and a photoreactive group whose bond is cleaved or whose structure is changed by a photoreaction, The immobilization material according to claim 12 , wherein the binding inhibition by the binding-inhibiting group is eliminated by light irradiation, thereby enabling the hydrophobic chain to bind to the biological substance.

14. The immobilization material according to claim 13, wherein the photoreactive group is a divalent group having a skeleton selected from the group consisting of a 2-nitrobenzyl skeleton, a coumarin-4-ylmethyl skeleton, a phenylcarbonylmethyl skeleton, a 7-nitroindolinocarbonyl skeleton, an azobenzene skeleton, a fulgide skeleton, a spiropyran skeleton, a spirooxazine skeleton, and a diarylethene skeleton.

15. The immobilization material according to claim 13 or 14, wherein the binding inhibitor group is a saturated or unsaturated hydrocarbon chain which may have a substituent.

16. the polymer has a photodegradable group in the molecule, The immobilization material according to any one of claims 1 to 15, wherein the hydrogel thin film layer can be decomposed by cleaving the photodegradable groups upon irradiation with light.

17. The immobilization material according to claim 16, wherein the photodegradable group is a divalent group having a skeleton selected from the group consisting of a 2-nitrobenzyl skeleton, a nitrophenylethyl ester skeleton, a coumarin-4-ylmethyl skeleton, a phenylcarbonylmethyl skeleton, a 7-nitroindolinocarbonyl skeleton, an azobenzene skeleton, a fulgide skeleton, a spiropyran skeleton, a spirooxazine skeleton, and a diarylethene skeleton.

18. The immobilization material according to any one of claims 1 to 17, wherein the biological material is selected from the group consisting of cells, organelles, vesicles, viruses, liposomes, and micelles.

19. An immobilization substrate for immobilizing a biological material having a lipid membrane on a surface, comprising:

19. A substrate for immobilization, having a surface modified with the immobilization material according to any one of claims 1 to 18, wherein the hydrogel thin film layer and the biological substance immobilization compound are present on the substrate in this order.

20. A method for producing a substrate for immobilizing a biological material, the surface of which is modified with the immobilization material according to any one of claims 1 to 18, comprising the steps of: The method includes the steps of: modifying the entire surface of a substrate with a hydrogel thin film layer made of a hydrogel having a structure in which polymer molecules are cross-linked with each other; and modifying the surface of the hydrogel thin film layer with a biological substance immobilization compound 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, wherein the biological substance immobilization compound has a concentration in the range of 3 to 2000 μM in solution.

21. When the biological material immobilization compound has a bond inhibitor that inhibits the bond between the biological material and the hydrophobic chain and a photoreactive group that is cleaved by a photoreaction or whose structure is changed, The manufacturing method according to claim 20, further comprising the step of patterning the surface of the immobilization substrate so that only specific regions on the substrate are endowed with the ability to bind to a biological substance, and irradiating the specific regions on the substrate surface with light to eliminate binding inhibition by the binding-inhibiting groups.

22. A step of contacting a solution containing a predetermined target biological substance with the immobilization substrate according to claim 19 to immobilize the target biological substance on the immobilization substrate; and A method for recovering a biological material, comprising the step of separating and recovering the immobilized target biological material from the immobilization substrate.

23. In the case where the polymer has a photodegradable group in the molecule, 23. The recovery method according to claim 22, comprising a step of irradiating a specific region of the immobilization substrate with light to decompose the hydrogel thin film layer, thereby separating and recovering only the target biological substance immobilized in the specific region from the immobilization substrate.

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