Surface-modified substrate for culturing and recovering cell clusters

Abstract

[Problem] The present invention addresses the problem of providing a novel technique capable of efficiently preparing cell clusters, such as spheroids and organoids, in a desired shape. [Solution] It has been found that a large amount of cell clusters, such as spheroids, having a fine shape can be prepared by using a culture substrate having a specific modified surface and obtained by laminating, on a substrate, a block layer that hinders adhesion to adherent cells, and then preferably patterning and modifying cell adhesion molecules having a photodegradable linker cleaved by irradiation with light.

Description

Surface-modified substrate for culturing and recovering cell aggregates

[0001] The present invention relates to a surface-modified substrate for culturing and recovering cell aggregates such as spheroids from adherent cells, a method for immobilizing and recovering the cell aggregates using the surface-modified substrate, and a method for producing the surface-modified substrate.

[0002] Technologies such as spheroids and organoids that mimic tissues in vitro are being actively studied in a wide range of fields, from basic research to understand complex in vivo developmental mechanisms and diseases to applications such as drug discovery, toxicity assessment, and tissue replacement therapy (Non-Patent Document 1). For example, in cancer treatment, it is believed that companion diagnostics using spheroids rather than cultured cells improves treatment outcomes, and techniques for rapidly preparing spheroids from patients' cancer cells have been widely reported. Furthermore, organoids from various organs are being prepared and utilized in new drug development. Spheroids and organoids are also expected to be used in a wide range of applications, including toxicity testing as an alternative to animal testing and regenerative medicine through tissue replacement.

[0003] However, current methods for producing spheroids and organoids rely on the stochastic generation of tissues through cell self-assembly, making it difficult to reproducibly form complex tissues (Non-Patent Document 2). This makes it difficult to uniformly analyze and accurately evaluate the effects of various factors on spheroid formation and the functions of the resulting products. Furthermore, for medical applications, it is necessary to prepare uniform spheroids with high reproducibility. Therefore, there is a need for a technology that can artificially arrange diverse cells and generate uniform spheroids with precisely controlled structure and shape.

[0004] In response to this, attempts to create cell aggregates such as spheroids and organoids have been reported, including inkjet printing (Non-Patent Document 3), photocurable gels (Non-Patent Document 4), microchannels (Non-Patent Document 5), methods using magnetic particles (Non-Patent Document 6), stimuli-responsive substrates (Non-Patent Documents 7 and 8), and soft lithography (Non-Patent Document 9).

[0005] However, these conventional methods have many issues, including the following. Specifically, 3D bioprinting techniques using inkjet printers or photocurable gels are still not easy to fabricate microstructures of 100 μm or less, and because they require the creation of numerous spheroids one by one, they require a significant amount of time for fabrication (Non-Patent Document 10). Techniques using microchannels are limited in the structures that can be fabricated, such as spherical or fibrous shapes. Techniques using magnetic particles have limitations in their application because the magnetic particles remain within the cells after fabrication. Furthermore, while techniques using photoresponsive culture substrates or soft lithography can prepare precise spheroids, they require the cells to be detached from the substrate by trypsin treatment or other methods when recovering them, making it impossible to maintain intercellular bonds during recovery.

[0006] On the other hand, to more accurately mimic the functions of spheroids in vivo and perform further analysis or medical applications after culture and evaluation, it is desirable to be able to recover precisely designed and prepared spheroids as they are. While a technique using temperature-responsive pNIPAM has been reported as such (e.g., Non-Patent Document 7), this technique does not allow for the preparation of spheroids with high spatial resolution at the level of a few cells. Therefore, currently, no technology has been established that allows for the mass preparation and recovery of precisely designed spheroids.

[0007] Hans H., Cell 2016, 165, 1586-1597; Sasai Y., Nature 2013, 493, 318-326Gjorevski N., et al., Science 2022, 375, eaaw9021Matsusaki M., et al., Adv. Health. Mater. 2013, 2, 534-539Ma X., et al., Pro. Nat. Acad. Sci. USA 2016, 113, 2206-2211Onoe H., et al., Nat. Mater. 2013, 12, 584-590Ino K., et al., Biotechnol. Bioeng. 2007, 97, 1309-1317Tsuda Y., et al., Biochem. Biophys. Res. Commun. 2006, 348, 937-944Kikuchi K., et al., Biotechnol. Bioeng. 2009, 103, 552-561Fukuda J., et al., Biomater. 2006, 27, 1479-1486Miri AK, et al., Lab Chip 2019, 19, 2019-2037

[0008] Therefore, an objective of the present invention is to provide a novel method that can efficiently prepare cell aggregates such as spheroids and organoids in desired shapes.

[0009] As a result of intensive research aimed at solving the above-mentioned problems, the inventors have discovered that it is possible to prepare large quantities of cell aggregates such as spheroids with precise shapes by using a culture substrate having a specific modified surface, which is formed by laminating a blocking layer that prevents adhesion of adherent cells on a substrate and then modifying the surface with cell adhesion molecules, preferably in a patterned form, that have a photodegradable linker that is cleaved by light irradiation. This finding led to the completion of the present invention. According to the present invention, multiple types of cells can be selectively arranged on a substrate by controlling light, and during culture, they can grow and organize only in the light-irradiated area to form cell aggregates with the desired shape. Furthermore, by irradiating the substrate with light again, cell aggregates can be detached and selectively recovered while maintaining intercellular bonds.

[0010] That is, in one aspect, the present invention relates to a surface-modified substrate for culturing and recovering adherent cells, and more specifically, <1> a surface-modified substrate for culturing and recovering adherent cells, comprising, on an upper portion of the substrate, a blocking layer that does not have adhesive properties to the adherent cells, a first modified layer modified with a first surface modifying agent on the upper portion of the blocking layer, and a second modified layer modified with a second surface modifying agent on the upper portion of the first modified layer, wherein the first surface modifying agent comprises a compound having the structure shown below: L-M-N (wherein L is a bonding moiety that bonds to the surface of the blocking layer; M is a hydrophilic linker; and N is a linking group that covalently bonds to an azide group or a thiol group); and the second surface modifying agent comprises a compound having the structure shown below: X-Y-Z (wherein X is a hydrophilic chain having an azide group or a thiol group at its terminal; Y is a photodegradable linker that is cleaved by irradiation with light; and Z is a cell attachment site that interacts with and binds to the adherent cells.<2> The surface-modified substrate according to the above <1>, which has a patterned surface modification in which the second modification layer is present only in a predetermined surface region of the first surface modifying agent; <3> The surface-modified substrate according to the above <1>, in which the block layer has a laminate structure of 4-branched or 8-branched polyalkylene glycol; <4> The surface-modifying agent according to the above <1>, in which the second surface modifying agent is a mixture of a compound having a cell-binding polypeptide as the cell attachment site Z and a compound having a hydrophobic chain as the cell attachment site Z; <5> The surface-modifying agent according to the above <4>, in which the molar ratio of the compound having a cell-binding polypeptide to the compound having a hydrophobic chain is in the range of 1000:1 to 1:1; <6> The surface-modified substrate according to the above <1>, in which the linking group N contains an alkynyl group or a group in which a protecting group has been introduced into an alkynyl group; <7> The surface-modified substrate according to the above <1>, in which the linking group N has a structure containing dibenzocyclooctyne (DBCO) or a precursor thereof; <8> The surface-modified substrate according to <1> above, wherein the substrate-binding portion L has a substituent capable of binding to the surface of the block layer by a covalent bond; <9> The surface-modified substrate according to <1> above, wherein the adherent cells after the culture are cell masses; <10> A surface-modified substrate for culturing and recovering adherent cells, the surface-modified substrate having, on an upper part of the substrate, a block layer that does not have adhesive properties to the adherent cells, and on an upper part of the block layer, a modification layer having a region to which the adherent cells selectively bind, the modification layer being formed with a surface modifying agent containing a compound having a cell-binding polypeptide and a compound having a hydrophobic chain, and the molar ratio of the compound having a cell-binding polypeptide to the compound having a hydrophobic chain is in the range of 1000:1 to 1:1; and <11> Provided is a surface-modified substrate for culturing and recovering adherent cells, the surface-modified substrate having, on an upper portion of the substrate, a blocking layer that does not have adhesive properties to the adherent cells, and, on an upper portion of the blocking layer, a modifying layer that has a region to which the adherent cells selectively bind, the blocking layer having a laminated structure in which 4-branched or 8-branched polyalkylene glycol is crosslinked.

[0011] In another aspect, the present invention also relates to a method for culturing and recovering adherent cells using the above-mentioned cell immobilization substrate, and more specifically, provides: <12> a method for culturing and recovering adherent cells, the method comprising the steps of bringing a solution containing predetermined target cells into contact with the surface-modified substrate according to any one of <1> to <11> above and immobilizing the target cells on the surface of the surface-modified substrate; culturing the immobilized target cells; and irradiating the surface-modified substrate with light to cleave a photodegradable linker present in the modification layer of the surface-modified substrate, thereby separating and recovering the immobilized target cells from the surface-modified substrate; and <13> the method according to <12> above, in which the separated and recovered target cells form cell clusters.

[0012] In a further aspect, the present invention also relates to a method for producing the above-mentioned cell immobilization substrate, and more specifically, <14> A method for producing a surface-modified substrate for culturing and recovering adherent cells, comprising: (A) a step of forming a block layer on an upper surface of a substrate, the block layer not having adhesive properties to the adherent cells; (B) a step of adding a first surface modifier onto the block layer to form a first modified layer, the first surface modifier comprising a compound having the structure shown below: L-M-N (wherein L is a bonding moiety that bonds to the surface of the block layer, M is a hydrophilic linker, and N is a linking group that covalently bonds to an azide group); and (C) a step of adding a second surface modifier onto the first modified layer, the second surface modifier comprising a compound having the structure shown below: X-Y-Z (wherein X is a hydrophilic chain having an azide group at its terminal, Y is a photodegradable linker that is cleaved by irradiation with light, and Z is a cell attachment site that binds to the adherent cells through interaction); (D) the production method comprising a step of forming a second modified layer on the first modified layer by forming a covalent bond between the linking group N in the first surface modifier and the azide group at the terminal of the hydrophilic chain X in the second surface modifier; <15> the production method according to the above <14>, which comprises, before the step (C), a step of irradiating a predetermined region on the surface of the first modified layer with light of a specific wavelength, thereby forming a patterned surface modification in which the second surface modifier is present only in the predetermined region; and <16> The manufacturing method according to the above item <14>, wherein the second surface modifying agent is a mixture of a compound having a cell-binding polypeptide as the cell attachment site Z and a compound having a hydrophobic chain as the cell attachment site Z, and the molar ratio of the compound having the cell-binding polypeptide to the compound having the hydrophobic chain is in the range of 1000:1 to 1:1.

[0013] According to the present invention, cell clusters (spheroids, organoids, etc.) composed of any cells can be mass-produced uniformly in a desired shape. Furthermore, multiple types of cells can be arranged in a desired pattern, making it possible to precisely prepare organoids that reproduce the functions of complex biological tissues. Furthermore, even after culturing, the present invention offers the advantage of being able to selectively recover cell clusters that maintain intercellular junctions by detaching them with light irradiation.

[0014] The method of the present invention can solve the problem of existing methods, which rely on the spontaneous aggregation of cells according to probability and are therefore unable to produce complex structures uniformly with good reproducibility.

[0015] Figure 1 is a schematic diagram showing the structure and layer structure of the compound used to form the blocking layer in Example 3. Figure 2 (A) shows an image of cells seeded on a surface coated twice with 4-branched PEG at a final concentration of 2.5 μM, before washing; (B) shows an image of the surface after washing; (C) shows an image of cells seeded on a surface coated six times with 4-branched PEG at a final concentration of 2.5 μM, before washing; and (D) shows an image of the surface after washing. Figure 3 is a graph showing the relationship between the number of blocking layer layers and the specific cell area occupied by cells after washing (three trials; error bars: standard deviation). Figure 4 (top) shows microscopic images of cells placed on a collagen-coated substrate modified with photoreactive molecules (images after 0, 1, and 2 days of culture); (middle) shows a similar image on a substrate modified with photoreactive molecules and linear PEG on the APTES surface; and (bottom) shows a similar image on a substrate modified with photoreactive molecules after 4-branched PEG was coated on the APTES surface. Figure 5 shows (A) a conceptual diagram of the process of detaching cells by light irradiation after placement (the cell adhesion molecule is cRGD); (B) a microscopic image after an array of rod-shaped spheroids has been generated (left), and a microscopic image after partial irradiation with light to detach the spheroids (middle), and after the spheroids in the light-irradiated area have been recovered by washing (right); (C) the size of the area irradiated to produce sheet-like spheroids; (D) a conceptual diagram of fixation for recovering spheroids detached by light; (E) a microscopic image after an array of sheet-like spheroids has been generated (left), and a microscopic image of the spheroids detached by light irradiation that have been recovered and transferred to a culture dish, observed from above (middle) and from the side (right).

[0016] 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.

[0017] 1. Surface-Modified Substrate A first aspect of the present invention is a surface-modified substrate for culturing and recovering adherent cells, characterized by having the following structure: (i) a blocking layer that is not adhesive to the adherent cells, provided on the top of the substrate; (ii) a first modified layer that is modified with a first surface modifier, provided on the top of the blocking layer, wherein the first surface modifier contains a compound having the structure "LM-N"; and (iii) a second modified layer that is modified with a second surface modifier, provided on the top of the first modified layer, wherein the second surface modifier contains a compound having the structure "X-Y-Z."

[0018] By having such a structure, the surface-modified substrate of the present invention can selectively immobilize adherent cells only in the region where the second modified layer having cell adhesion sites on its surface is present, and by culturing the cells, cell aggregates such as spheroids and organoids can be formed, and then the cell aggregates can be selectively recovered by light irradiation.

[0019] In the present invention, "adherent cells" generally refer to cells that need to adhere to an appropriate surface for proliferation, and are also called adherent cells or anchorage-dependent cells. Adherent cells are not particularly limited as long as they are capable of forming cell masses such as spheroids and organoids. Examples of such cells include various cultured cell lines such as Chinese hamster ovary-derived CHO cells, mouse connective tissue L929 cells, human fetal lung-derived normal diploid fibroblasts (TIG-3 cells), human fetal kidney-derived cells (HEK293 cells), human cervical cancer-derived HeLa cells, Vero cells, and NIH3T3 cells, as well as epithelial cells and endothelial cells that constitute various tissues and organs in vivo; Examples of such cells include contractile skeletal muscle cells, smooth muscle cells, and cardiac muscle cells; neuronal cells, glial cells, and fibroblasts that constitute the nervous system; hepatic parenchymal cells, non-hepatic parenchymal cells, and adipocytes that are involved in the metabolism of living organisms; induced pluripotent stem (iPS) cells, embryonic stem (ES) cells, embryonic germ (EG) cells, embryonic carcinoma (EC) cells, mesenchymal stem cells, liver stem cells, pancreatic stem cells, skin stem cells, muscle stem cells, and germline stem cells, as well as various stem cells and progenitor cells of various tissues, and cells induced to differentiate from such stem cells and progenitor cells. Other examples include cells (living cells) contained in blood, lymph, cerebrospinal fluid, sputum, urine, or feces, as well as microorganisms, viruses, and protozoa present in the body or in the environment.

[0020] The elements (i) to (iii) that constitute the surface-modified base material of the present invention will be described below.

[0021] 1-1. Blocking Layer The blocking layer in the present invention is a layer that does not have adhesive properties with adherent cells and has the function of blocking the attachment of adherent cells to the substrate surface. When the target cells to be immobilized on the substrate surface are suspension cells, surface modification with a compound having a cell attachment site, as described below, allows selective attachment only to areas where such a compound is present. However, due to their own adhesive properties, adherent cells also non-selectively attach to substrate surfaces where such a compound having a cell attachment site is not present, making it impossible to control their immobilization on the substrate surface. For this reason, such a blocking layer is provided in the present invention.

[0022] The blocking layer in the present invention is not particularly limited as long as it is a material to which adhesive cells do not adhere, but from the viewpoint of ease of modification of the substrate surface, etc., hydrophilic polymers such as polyalkylene glycols or biocompatible polymers can typically be used. Among such hydrophilic polymers and biocompatible polymers, polyalkylene glycols are preferred.

[0023] The weight-average molecular weight of the polyalkylene glycol used is preferably 500 to 100,000, and more preferably 2,000 to 40,000. The weight-average molecular weight is a value measured by MALDI-TOF-MS.

[0024] Furthermore, such a block layer preferably has a structure in which multiple polyalkylene glycols are laminated. Such a laminate structure can be composed of, for example, two or more layers in which a first polyalkylene glycol layer having one or more nucleophilic functional groups at the side chain or terminal and a second polyalkylene glycol layer having one or more electrophilic functional groups at the side chain or terminal are alternately laminated. In this case, the first and second polyalkylene glycols can be linked to each other by crosslinking. Furthermore, since unreacted nucleophilic or electrophilic functional groups remain even after the first and second polyalkylene glycols are linked by crosslinking, these can be used to bond with the binding entity L in the first surface modifier.

[0025] Preferably, a gel can be formed by crosslinking between polyalkylene glycols. In this specification, "gel" broadly means a dispersion system of polymers that has high viscosity and has lost fluidity, and "hydrogel" means a gel containing water as a solvent (dispersion medium). Hydrogels having a network structure, particularly a three-dimensional network structure, are preferred.

[0026] In a preferred embodiment, the polyalkylene glycol forming the blocking layer is polyethylene glycol (PEG), and 4- or 8-branched PEG is particularly preferred. The inventors have found that such branched PEG has excellent blocking properties against the attachment of adherent cells to a substrate.

[0027] It is known that four-branched polyethylene glycol (Tetra-PEG) forms a gel with a network structure (Tetra-PEG) through an AB cross-end coupling reaction between two four-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 two polymer solutions, and the gelation time can be controlled by adjusting the pH and ionic strength during gel preparation.

[0028] Nucleophilic functional groups in polyalkylene glycols 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, -CO ２ PhNO ２ (Ph represents an o-, m-, or p-phenylene group), or a nitrophenyl group, and those skilled in the art can appropriately use other known active ester groups. Preferably, the nucleophilic functional group is an amino group, and the electrophilic functional group is an N-hydroxy-succinimidyl (NHS) group.

[0029] In addition to such combinations 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 an azide group and an alkyne group (cycloaddition reaction), an azide group and a dibenzocyclooctyne group (cycloaddition 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, or a ketone group and an aminooxy group.

[0030] The blocking layer is formed on the substrate, and "on the substrate" here means that the blocking layer is located above the substrate (i.e., in the direction of the first modification layer). Therefore, the blocking layer may be formed directly on the substrate surface, or a coating layer may be provided on the substrate surface and the blocking layer may be formed on the surface of the coating layer. Examples of such coating layers include collagen, bovine serum albumin (BSA), 3-aminopropyltriethoxysilane (APTES), and ovalbumin.

[0031] In a preferred embodiment, when a layer of a first polyalkylene glycol having one or more nucleophilic functional groups in the chain or at the end and a layer of a second polyalkylene glycol having one or more electrophilic functional groups in the side chain or at the end are laminated, such a laminate can be formed by first adding a solution containing the first polyalkylene glycol to the surface of the substrate or the coating layer, and then adding a solution containing the first polyalkylene glycol.

[0032] The material and shape of the substrate used in 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 (e.g., particulate or colloidal materials 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, or treated with a silane coupling agent having a substituent introduced into the substrate surface, in order to introduce amino groups, carboxyl groups, hydroxyl groups, or the like, or reactive functional groups may be introduced by plasma treatment.

[0033] 1-2. First Surface Modifier The first surface modifier forming the first modification layer in the present invention has three moieties: a moiety for bonding to the surface of the block layer, a hydrophilic chain moiety, and a moiety for linking with an azide group or a thiol group in the second surface modifier. Specifically, the first surface modifier includes a compound having the structure shown below: L-M-N

[0034] In the formula, L is a linking moiety that binds to the surface of the blocking layer; M is a hydrophilic linker; and N is a linking group that covalently bonds with the azide or thiol group.

[0035] <Bonding Moiety L> The bonding moiety L preferably has a substituent capable of bonding to the surface of the block layer by a covalent bond. As such a substituent, an active ester group such as N-hydroxysuccinimide (NHS), a carboxyl group, a silanol group, a disulfide group, or a thiol group can be preferably used.

[0036] In an embodiment in which the block layer contains a polyalkylene glycol having one or more nucleophilic functional groups or electrophilic functional groups at the side chain or end, the bonding portion L is preferably a nucleophilic functional group or electrophilic functional group corresponding to the polyalkylene glycol.

[0037] Alternatively, the first surface modifier may be immobilized on a block layer by reacting a polypeptide or nucleic acid with a block layer having a substance on its surface that can bind to the polypeptide or nucleic acid as the binding moiety L. For example, a combination of complementary DNA strands, such as a combination of biotin and avidin, may be used.

[0038] <Hydrophilic Linker M> The hydrophilic linker M is preferably composed of a hydrophilic polymer chain. Examples of such hydrophilic polymers include polysaccharides such as polyalkylene glycol, polyvinyl alcohol, polyacrylic acid, polypeptides, 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, with an average polymerization number in the range of 2 to 500 (preferably 45 to 500). The hydrophilic polymer is preferably a biocompatible polymer, and more preferably polyethylene glycol (PEG). The hydrophilic chain M may further have an optional substituent.

[0039] More specifically, the hydrophilic linker M preferably has the following partial structure having a repeating unit derived from ethylene glycol:

[0040] Here, m is a natural number greater than 2, and preferably a natural number from 3 to 100. More preferably, m is a natural number from 4 to 40.

[0041] <Linking Group N> Any functional group known in the art can be used as the linking group N, as long as it is capable of covalently bonding to an azide group or a thiol group in the second surface modifier. In this way, when the second surface modifier is added to the substrate surface formed with the first surface modifier, the second surface modifier reacts with the azide group or the thiol group in the molecule, thereby covalently linking the second surface modifier to the upper layer of the first surface modifier.

[0042] Typically, the linking group N can be an alkynyl group, a group in which a protecting group has been introduced into an alkynyl group, or a functional group or partial structure containing such a group. In this case, the protecting group is not particularly limited, but examples thereof include cyclic ketones such as cyclopropenone.

[0043] The partial structure containing such an alkynyl group or a group in which a protecting group has been introduced into an alkynyl group is preferably a structure containing dibenzocyclooctyne (DBCO) or a precursor thereof. Furthermore, the precursor of DBCO may include a structure in which the alkynyl group of DBCO is protected by providing a protecting group. DBCO may have any substituent.

[0044] DBCO may have any substituent. 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. The substituent in DBCO is not particularly limited, but is preferably an alkoxy group such as a methoxy group.

[0045] Non-limiting examples of linking groups N that include DBCO or precursors thereof include the following structures: (In the formula, R １ represents the point of attachment to the hydrophilic chain M.)

[0046] The linkage between each of the above L, M, and N sites can be, for example, a covalent bond such as an amide bond, an ester bond, an ether bond, a thioether bond, a carbamate bond, a thiocarbamate bond, a triazole bond, a urea bond, etc. The linkage between the binding site L and the hydrophilic linker M, and the linkage between the hydrophilic linker M and the linking group N may be the same or different bonding modes.

[0047] In one embodiment, an optional linker may further exist between each of the L, M, and N moieties. Such a linker is not particularly limited, but may be, for example, C 6-14 arylene group or C 1-10 The alkylene group is an alkylene group. Here, the 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 may be substituted with NH, N(C 1-10 It may be replaced by alkyl), O or S. In some cases, it is preferred that the bond between L and M and the bond between M and N each independently have a structure selected from the group consisting of an alkylene structure, an amide structure, an ester structure, an amino structure, and an ether structure.

[0048] In this specification, the term "alkyl or alkyl group" may be any of a linear, branched, or cyclic aliphatic hydrocarbon groups, or a combination thereof. The number of carbon atoms in the alkyl group is not particularly limited, but for example, alkyl groups having 1 to 20 carbon atoms (C １～２０ ), carbon number 1 to 15 (C １～１５ ), carbon number 1 to 10 (C １～１０ In this specification, the alkyl group may have one or more optional substituents. For example, C １～８Alkyl includes methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neo-pentyl, n-hexyl, isohexyl, n-heptyl, n-octyl, and the like. Examples of the substituent include, but are not limited to, an alkoxy group, a halogen atom (which may be a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom), an amino group, a mono- or di-substituted amino group, a substituted silyl group, or an acyl. When an alkyl group has two or more substituents, these may be the same or different. The same applies to the alkyl moiety of other substituents containing an alkyl moiety (e.g., an alkoxy group, an arylalkyl group, and the like).

[0049] In the present specification, "alkylene" refers to a divalent group consisting of a linear or branched saturated hydrocarbon, and examples thereof include methylene, 1-methylmethylene, 1,1-dimethylmethylene, ethylene, 1-methylethylene, 1-ethylethylene, 1,1-dimethylethylene, 1,2-dimethylethylene, 1,1-diethylethylene, 1,2-diethylethylene, 1-ethyl-2-methylethylene, trimethylene, 1-methyltrimethylene, 2-methyltrimethylene, 1,1-dimethyltrimethylene, 1,2 2-dimethyltrimethylene, 2,2-dimethyltrimethylene, 1-ethyltrimethylene, 2-ethyltrimethylene, 1,1-diethyltrimethylene, 1,2-diethyltrimethylene, 2,2-diethyltrimethylene, 2-ethyl-2-methyltrimethylene, tetramethylene, 1-methyltetramethylene, 2-methyltetramethylene, 1,1-dimethyltetramethylene, 1,2-dimethyltetramethylene, 2,2-dimethyltetramethylene, 2,2-di-n-propyltrimethylene, and the like.

[0050] As used herein, "amide or amido group" includes both RNR'CO-- (when R=alkyl, alkylaminocarbonyl-) and RCONR'-- (when R=alkyl, alkylcarbonylamino-).

[0051] Specific examples of the first surface modifier used in the present invention include Compound 1 and Compound 2 having the following structures, but are not limited to these. (In the formula, n is a natural number from 1 to 500.)

[0052] 1-3. Second Surface Modifier The second surface modifier forming the second modified layer in the present invention has three moieties: a hydrophilic chain having an azide group or a thiol group for bonding to the surface of the first modified layer, a photodegradable linker that is cleaved by irradiation with light, and a cell attachment moiety that can selectively bind to adherent cells. Specifically, the second surface modifier includes a compound having the structure shown below: X-Y-Z

[0053] In the formula, X is a hydrophilic chain having an azide group or a thiol group at the end; Y is a photodegradable linker that is cleaved by irradiation with light; and Z is a cell attachment site that interacts with and binds to the adherent cells.

[0054] <Hydrophilic Chain X> The hydrophilic chain X has an azide group or a thiol group at its terminal, which can be covalently linked to the linking group N in the first surface modifier. The hydrophilic chain in X is preferably composed of a hydrophilic polymer. Such hydrophilic polymers can be the same as those exemplified for the hydrophilic linker M above. Preferably, both the hydrophilic linker M and the hydrophilic chain X are hydrophilic polymers.

[0055] <Photodegradable Linker Y> The photodegradable linker Y contains a functional group that can be decomposed by irradiation with light such as visible light or ultraviolet light. This allows target adherent cells to be captured and cultured at the cell attachment site Z described below, and then cell masses such as spheroids or organoids formed from the target cells to be separated and recovered from the surface of the substrate by irradiating the cell with light.

[0056] The functional group in the photodegradable linker Y that can be decomposed by irradiation with light is not particularly limited as long as it can cleave the hydrophilic chain X and the cell attachment site Z by a photoreaction, but examples of divalent groups 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. Preferably, the divalent group has a 2-nitrobenzyl skeleton.

[0057] <Cell attachment site Z> The cell attachment site Z is a site that interacts with and binds to adherent cells to capture and immobilize the adherent cells. Typically, the cell attachment site Z is a cell-binding polypeptide or a hydrophobic chain.

[0058] In a preferred embodiment, the second surface modifying agent can be a mixture of a compound having a cell-binding polypeptide as the cell attachment site Z and a compound having a hydrophobic chain as the cell attachment site Z. In this case, the molar ratio of the compound having the cell-binding polypeptide to the compound having the hydrophobic chain is preferably in the range of 1000:1 to 1:1, more preferably 100:1 to 1:1, and even more preferably 100:1 to 10:1. By using such a mixture, the adherent cells can be guided to a desired position by the hydrophobic chain, while the adherent cells can be firmly immobilized by the cell-binding polypeptide such as an RGD peptide.

[0059] The cell-binding polypeptide can be a known polypeptide molecule or protein capable of binding to target adherent cells, depending on the type of target cell to be immobilized. Typical examples of such proteins include lectins and antibodies. Other proteins capable of binding to cell surfaces include cell adhesion proteins such as collagen, fibronectin, vitronectin, and laminin; and proteins involved in tight junctions such as E-cadherin. Furthermore, the polypeptide molecule may include peptides with partial structures of cell adhesion proteins, such as cRGD peptides and polyarginine peptides, or cationic cell-penetrating peptides. Preferably, the cell-binding polypeptide is an RGD polypeptide or a cyclic RGD polypeptide.

[0060] As used herein, the term "lectin" refers to a protein that recognizes the partial structure, entire structure, or glycopeptide portion of a sugar chain bound to a complex carbohydrate such as a glycoprotein, glycolipid, proteoglycan, glycopeptide, lipopolysaccharide, peptidoglycan, or glycoside such as a steroid compound, and specifically binds to the sugar chain. Examples of lectins include plant lectins, fungal lectins, animal lectins, cytokines with sugar-binding activity, GAG-binding proteins, microbial adhesins, bacterial toxins, and viral hemagglutinins. Lectins may be naturally occurring or artificially synthesized.

[0061] Preferably, the lectin used in the present invention is BC2LCN lectin or a modified form thereof. BC2LCN lectin is the N-terminal domain of the BC2L-C protein derived from the gram-negative bacterium Burkholderia cenocepacia. A recombinant protein (rBC2LCN lectin) obtained by expressing this BC2LCN lectin in Escherichia coli can be preferably used. rBC2LCN lectin is an undifferentiation marker that has high affinity for mucin-like O-glycans specific to the surface of human ES cells and human iPS cells. The rBC2LCN lectin can be mass-produced using transformed bacteria, and specific preparation methods are described, for example, in International Publication WO 2016 / 147514. BC2LCN lectin or a modified form thereof also includes BC2LCN lectin modified with any tag peptide known in the art.

[0062] Furthermore, the hydrophobic chains used as the cell attachment site Z can bind to and capture adherent cells through non-covalent interactions. Specifically, the hydrophobic chains can bind to adherent cells through hydrophobic interactions with lipid moieties in the cell membrane, which is a lipid bilayer membrane.

[0063] Such hydrophobic chains are not particularly limited as long as they can bind to adherent cells through hydrophobic interactions, and may be saturated or unsaturated hydrocarbon chains which may have a substituent. Examples of such hydrocarbon chains include, for example, C 7-30 Alkyl group (preferably C 7-22 alkyl 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 an aryl group (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. 7-30an alkyl group, a C group 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 the phospholipids that constitute cell membranes, is preferred. Furthermore, these hydrophobic chains may be substituted with any substituent and may contain heteroatoms such as N, S, and O.

[0064] The linkage between each of the above X, Y, and Z sites can be, for example, a covalent bond such as an amide bond, an ester bond, an ether bond, a thioether bond, a carbamate bond, a thiocarbamate bond, a triazole bond, or a urea bond. The linkage between each site may be the same or different bonding modes.

[0065] In one embodiment, an optional linker may further exist between each of the X, Y, and Z sites. Such a linker is not particularly limited, but may be, for example, C 6-14 arylene group or C 1-10 The alkylene group is an alkylene group. Here, the 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 may be substituted with NH, N(C 1-10It may be replaced by alkyl), O or S. In some cases, it is preferred that the bond between L and M and the bond between M and N each independently have a structure selected from the group consisting of an alkylene structure, an amide structure, an ester structure, an amino structure, and an ether structure.

[0066] As described below, after modifying the entire surface of the substrate with a first modification layer, specific regions can be irradiated with UV light to pattern the second modification layer so that it is present only in desired surface regions.

[0067] In another preferred embodiment, the surface-modified substrate of the present invention can be characterized by having the following structure: a blocking layer on top of the substrate that does not have adhesive properties to the adherent cells; and a modified layer on top of the blocking layer that has an area to which the adherent cells selectively bind, the modified layer being formed from a surface modifying agent containing a compound having a cell-binding polypeptide and a compound having a hydrophobic chain, and the molar ratio of the compound having a cell-binding polypeptide to the compound having a hydrophobic chain is in the range of 1000:1 to 1:1.

[0068] Preferably, in the compound of the modifying layer, a combination can be used in which the cell-binding polypeptide is an RGD polypeptide or a cyclic RGD polypeptide, and the hydrophobic chain is a saturated or unsaturated hydrocarbon chain which may have a substituent.

[0069] In yet another preferred embodiment, the surface-modified substrate of the present invention can also be characterized by having the following structure: a block layer that does not have adhesive properties to the adherent cells on top of the substrate, and a modified layer that has an area to which the adherent cells selectively bind on top of the block layer, and the block layer has a laminate structure in which 4-branched or 8-branched polyalkylene glycol is crosslinked.

[0070] Preferably, the laminated structure comprises two or more layers in which a layer of a first polyalkylene glycol having one or more nucleophilic functional groups at its side chain or terminal and a layer of a second polyalkylene glycol having one or more electrophilic functional groups at its side chain or terminal are alternately laminated, and the first and second polyalkylene glycols are linked to each other by crosslinks. The types of polyalkylene glycols that can be used here are as described above for the block layer.

[0071] 2. Method for Producing a Cell Immobilization Substrate of the Present Invention A second aspect of the present invention is a method for producing the above-mentioned surface-modified substrate for culturing and recovering adherent cells, characterized in that it comprises the following steps (A) to (D): (A) a step of forming a block layer on top of a substrate, which does not have adhesiveness to the adherent cells; (B) a step of adding a first surface modifier onto the block layer to form a first modified layer, wherein the first surface modifier contains a compound having the structure shown below: L-M-N (wherein L is a bonding moiety that bonds to the surface of the block layer, M is a hydrophilic linker, and N is a linking group that covalently bonds to an azide group); (C) a step of adding a second surface modifier onto the first modified layer, wherein the second surface modifier contains a compound having the structure shown below: X-Y-Z (wherein X is a hydrophilic chain having an azide group at its terminal, Y is a photodegradable linker that is cleaved by irradiation with light, and Z is a cell attachment site that interacts with and binds to the adherent cells); (D) a step of forming a second modified layer on top of the first modified layer by forming a covalent bond between the linking group N in the first surface modifier and the azide group at the terminal of the hydrophilic chain X in the second surface modifier.

[0072] Specifically, in step (A), the surface of the substrate is first modified with a blocking layer that does not have adhesive properties to adherent cells and thus functions to inhibit the attachment of adherent cells to the substrate surface. Next, in step (B), a first surface modifier is added to modify the substrate surface, and in step (C), a second surface modifier is further added to the substrate, thereby performing surface modification of the substrate in two steps. Here, the first surface modifier contains a linking group N capable of covalently bonding to an azide group or a thiol group within its molecule. When the second surface modifier is added, it reacts with the azide group or thiol present in the hydrophilic chain X of the second surface modifier. In step (D), the second surface modifier is covalently linked to the top layer of the first surface modifier, thereby obtaining a surface-modified substrate having the second surface modifier on the outermost surface. While adherent cells cannot adhere to the blocking layer, the second surface modifier contains cell attachment sites capable of binding to adherent cells, allowing them to selectively capture and immobilize adherent cells only at the cell attachment sites on the substrate surface.

[0073] Each step in the production method of the present invention will be specifically described below.

[0074] 2-1. Step (A) Step (A) is a step of forming a block layer on the substrate that does not have adhesive properties to adherent cells. As described above, the material for forming such a block layer is not particularly limited as long as it is a material to which adherent cells do not adhere. However, from the viewpoint of ease of modification of the substrate surface, hydrophilic polymers such as polyalkylene glycols or biocompatible polymers can typically be used. Among such hydrophilic polymers and biocompatible polymers, polyalkylene glycols are preferred. Details of the polyalkylene glycols used are as described above.

[0075] Such a block layer preferably has a structure in which multiple polyalkylene glycols are laminated. Such a laminate structure can be composed of, for example, two or more layers in which a first polyalkylene glycol layer having one or more nucleophilic functional groups at the side chain or terminal and a second polyalkylene glycol layer having one or more electrophilic functional groups at the side chain or terminal are alternately laminated. In this case, the first and second polyalkylene glycols can be linked to each other by crosslinking. Furthermore, since unreacted nucleophilic or electrophilic functional groups remain even after the first and second polyalkylene glycols are linked by crosslinking, these can be used to bond with the binding entity L in the first surface modifier.

[0076] As described above, "above the substrate" in step (A) means being located above the substrate (i.e., in the direction of the first modification layer). Therefore, the blocking layer may be formed directly on the substrate surface, or an optional coating layer may be provided on the substrate surface, and the blocking layer may be formed on the surface of the coating layer. Examples of such coating layers that can be used include collagen, bovine serum albumin (BSA), 3-aminopropyltriethoxysilane (APTES), and ovalbumin.

[0077] Specifically, the block layer is formed by contacting a solution containing a hydrophilic polymer or a biocompatible polymer with the surface of the substrate or coating layer. The type of solvent in the polymer solution is not particularly limited, but examples include buffers such as phosphate buffer, borate buffer, Tris buffer, acetate buffer, carbonate buffer, and Good's buffer, or isotonic solutions thereof; organic solvents such as acetonitrile, dimethyl sulfoxide, and dimethylformamide; and mixtures of the buffers or isotonic solutions with the organic solvents. To avoid damaging or denaturing the substrate, the polymer may be dissolved in an organic solvent (e.g., acetonitrile, dimethyl sulfoxide, or dimethylformamide) in which it is easily soluble, and then sufficiently diluted with the buffer or isotonic solution. The temperature at which the polymer solution is contacted with the substrate or coating layer is not particularly limited, but is preferably, for example, −78 to 200°C, and more preferably 0 to 100°C. The contact time is typically about 1 minute to 72 hours, with 30 minutes to 24 hours being preferred. After modification, the substrate surface is preferably washed with water.

[0078] In a preferred embodiment, when a layer of a first polyalkylene glycol having one or more nucleophilic functional groups in the chain or at the end is laminated with a layer of a second polyalkylene glycol having one or more electrophilic functional groups in the side chain or at the end, such a laminate can be formed by first adding a solution containing the first polyalkylene glycol to the surface of the substrate or the coating layer, and then adding a solution containing the second polyalkylene glycol.

[0079] 2-2. Step (B) As described above, step (B) is a step of adding a first surface modifier (LM-N) to the entire or part of the surface of the blocking layer, thereby forming a first modified layer. The first modified layer can be considered to be a base layer for connecting a second surface modifier in a later step. The first surface modifier is preferably arranged in the form of a monolayer on the surface of the substrate. The types and preferred embodiments of the first surface modifier that can be used are as described above.

[0080] Typically, the first modification layer is formed by contacting the surface of the block layer with a solution containing a first surface modifier. As in the case of the block layer, the type of solvent in the solution containing the first surface modifier is not particularly limited, and examples thereof include buffers such as phosphate buffer, borate buffer, Tris buffer, acetate buffer, carbonate buffer, and Good's buffer, or isotonic solutions thereof; organic solvents such as acetonitrile, dimethyl sulfoxide, and dimethylformamide; and mixtures of the buffers or isotonic solutions with the organic solvents. Alternatively, the first surface modifier may be dissolved in an organic solvent (e.g., acetonitrile, dimethyl sulfoxide, or dimethylformamide) in which it is easily soluble, and then sufficiently diluted with the buffer or isotonic solution to be used.

[0081] The temperature at which the solution is added is not particularly limited, but is preferably, for example, −78 to 200° C., more preferably 0 to 100° C. The contact time between the solution and the substrate is usually about 1 minute to 72 hours, preferably 30 minutes to 24 hours. After modification, it is preferable to wash the surface of the substrate with water.

[0082] 2-3. Step (C) As described above, step (C) is a step of further adding a second surface modifier (X-Y-Z) to the first modified layer. The second surface modifier contains a hydrophilic chain X having an azide group or a thiol group at its terminal for linking to the linking group N in the first surface modifier, and a cell attachment site Z that interacts with and binds to adherent cells.

[0083] As described above, the cell attachment site Z is typically a cell-binding polypeptide or a hydrophobic chain. The types and preferred embodiments of the second surface modifier that can be used have been described above. In a preferred embodiment, the second surface modifier can be a mixture of a compound having a cell-binding polypeptide as the cell attachment site Z and a compound having a hydrophobic chain as the cell attachment site Z. By using such a mixture, the adherent cells can be guided to a desired position by the hydrophobic chain, while the adherent cells can be firmly fixed by the cell-binding polypeptide such as an RGD peptide.

[0084] The second surface modifier can be added to the substrate by contacting the substrate with a solution containing the second surface modifier. The conditions for the solution, such as the solvent and contact time, are the same as those for the first surface modifier.

[0085] 2-4. Step (D) Step (D) is a step of covalently linking the second surface modifier to the upper layer of the first modification layer by reacting the linking group N in the first surface modifier with the azide group or thiol in the second surface modifier, thereby obtaining a surface-modified substrate having cell attachment sites Z in the second surface modifier on the outermost surface.

[0086] In a preferred embodiment of the production method of the present invention, a surface-modified substrate can be provided that has been modified with a second surface modifier having cell attachment sites Z at desired locations using a photopatterning technique. This can be achieved by using, as the linking group N in the first surface modifier, a functional group that becomes reactive with an azide group or a thiol group upon light irradiation, thereby linking the second surface modifier only to the light-irradiated region.

[0087] More specifically, the production method of the present invention can further include a step of irradiating predetermined regions of the first modified layer with light of a specific wavelength before step (C), thereby obtaining a patterned surface modification in which the second surface modifier is present only in the predetermined surface regions.

[0088] For example, as shown below, when a DBCO precursor having a protecting group attached thereto is used as the linking group N in the first surface modifier, DBCO is generated when the first surface modifier is irradiated with light of 360 nm, and this generates a structure that can bond to an azide group or a thiol group of the second surface modifier by a Huisgen cycloaddition reaction under physiological conditions. (In the formula, R １ represents the point of attachment to the hydrophilic chain M.)

[0089] This allows the second surface modifier to be modified only in the area irradiated with light, and by setting the light irradiation area according to the desired pattern, it becomes possible to capture and immobilize adherent cells only in the specific patterned area.

[0090] The wavelength of the light to be irradiated may be determined depending on the type of linking group N, and light having a wavelength in the range of 157 to 600 nm, preferably around 250 to 450 nm, is typically irradiated. The light may be ultraviolet light. Examples of light sources that can be used include sunlight, electric light such as a mercury lamp, laser light (semiconductor laser, solid-state laser, gas laser), light emitted from a light-emitting diode, and light emitted from an electroluminescent element. Regarding the light irradiation method, light from a light source may be uniformly irradiated onto the substrate surface through an appropriate filter as necessary, or a so-called photomask may be used to perform pattern exposure in a desired shape. Alternatively, light may be focused using a lens or mirror and irradiated onto a fine shape. Alternatively, the focused light beam may be used for scanning exposure.

[0091] In the case of pattern exposure, contact exposure, which is an exposure method in which a photomask and a substrate are brought into contact with each other, may be used. Alternatively, proximity exposure, which is a non-contact exposure method in which the gap between the photomask and the substrate is set to a few μm to a few tens of μm, may be used. Furthermore, a projection exposure method (maskless exposure method) in which an image created by a liquid crystal or digital mirror device is projected onto the work surface may be used. The energy of light irradiation may be sufficient as long as the substrate surface modified with the photoresponsive cell immobilization agent can exhibit the function of immobilizing cells, and is typically 0.001 to 1000 J / cm. ２ and 0.01 to 100 J / cm ２ is preferred.

[0092] The shape of the pattern is not particularly limited, but may be, for example, a pattern in which cells can be immobilized at regular intervals in the horizontal direction (X direction) and / or the vertical direction (Y direction). In this case, there is no limit to the number of locations at which cells can be immobilized, and even a single location is included in the term "patterning."

[0093] 2. Cultivation and Recovery Method of the Present Invention In a further aspect, the present invention also relates to a method for culturing and recovering adherent cells using the above-described surface-modified substrate.

[0094] More specifically, the culture and recovery method of the present invention includes the steps of bringing a solution containing predetermined target cells into contact with the surface-modified substrate to immobilize the target cells on the surface of the surface-modified substrate; culturing the immobilized target cells; and separating and recovering the immobilized target cells from the surface-modified substrate by irradiating the surface-modified substrate with light to cleave a photodegradable linker present in the modification layer of the surface-modified substrate.

[0095] As described above, in conventional methods, when the target cells to be immobilized are adhesive cells, their own adhesive properties cause them to non-selectively adhere to substrate surfaces that do not contain compounds having such cell attachment sites, making it impossible to control their immobilization on the substrate surface. Therefore, in the present invention, a blocking layer that functions to inhibit adhesion of adhesive cells to the substrate surface is provided, and a second modification layer having cell attachment site Z is provided on the outermost surface, making it possible to selectively immobilize adhesive cells only in the desired region.

[0096] To perform these methods, a cell immobilization substrate can be placed in a microchannel. The target cells are adherent cells, the types of which are as described above. Typically, the adherent cells immobilized on the surface-modified substrate form cell aggregates such as spheroids or organoids through culture. Therefore, the separated and recovered target cells are preferably cell aggregates such as spheroids or organoids.

[0097] After immobilization, the target cells can be cultured using techniques and culture conditions commonly used in the art.

[0098] The sugar solution and enzyme solution used in cell separation and recovery competitively weaken the bond between the target cells immobilized on the substrate and the cell attachment site Z, thereby releasing the target cells from the substrate surface under mild conditions. An example of the sugar solution is a fucose solution, and examples of the enzyme solution are a cell detachment solution such as Accutase (registered trademark), a trypsin / EDTA solution, and a protease solution.

[0099] In a preferred embodiment, as described above, the second modification layer is photopatterned to allow immobilization and cultivation of target cells in specific regions.

[0100] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0101] Example 1: Synthesis of DBCO-containing surface modifier (first surface modifier) ​​The following compounds 1 and 2 were synthesized, each of which has dibenzocyclooctyne (DBCO) as a linking group capable of covalently bonding to an azide group or a thiol group in a second surface modifier. These compounds correspond to the first surface modifier. (wherein n is 73 on average.)

[0102] <Synthesis of Compound 1> Compound 1 was synthesized according to the following synthesis scheme: A reactive group for modifying the substrate surface and a DBCO group that binds to an azide group by a Huisgen cycloaddition reaction were introduced to both ends of a hydrophilic polymer (polyethylene glycol: PEG) that can suppress nonspecific cell adhesion.

[0103] [Compound 4] A 100 mL three-necked recovery flask was heated and dried with a heat gun. Magnesium (2.4 g, 98.7 mmol, 13.1 eq) was added and purged with Ar. 5 mL of dry THF and 1,2-dibromoethane (100 μL, 1.17 mmol, 0.16 eq) were added. 3-methoxybenzyl chloride (3.0 mL, 21 mmol, 2.7 eq) and 30 mL of dry THF were added to a dropping funnel connected to the three-necked recovery flask, dried, and purged with Ar. The mixture was added dropwise over 3.5 hours with stirring in an ice bath. The reaction solution was then transferred to a 100 mL two-necked recovery flask containing compound 3 (0.92 mL, 7.52 mmol, 1 eq) after drying and purging with Ar. The mixture was then refluxed at 71°C for 2 hours. TLC (chloroform, Rf: Compound 3 = 0.66, imine intermediate = 0.51) confirmed the disappearance of Compound 3. NaBH (1.6016 g, 42.34 mmol, 5.63 eq) was added, the reaction solution was transferred to a 100 mL two-necked recovery flask, dried up, purged with Ar, and charged with 30 mL of dry MeOH, and stirred.

[0104] After 48 hours, the completion of the reaction was confirmed by TLC (Hexane / Acetone = 2 / 1, Rf: Imine = 0.63, Amine = 0). After distilling off the solvent, 30 mL of pure water was added and the mixture was extracted three times with 50 mL of CHCl. ​​At this point, the aqueous phase became an emulsion, so it was filtered through Celite. NaSO was added to the extracted organic phase to dry it, and the mixture was suction filtered and the solvent was distilled off. Succinic anhydride (0.9641 g, 9.634 mmol, 1.28 eq) was placed in a beaker and dissolved in 20 mL of acetonitrile. This was then added to the extracted organic layer. DMAP (0.1915 g, 1.567 mmol, 0.21 eq) was then added. After about two days, we attempted to confirm the disappearance of the raw material (amine) by TLC, but the DMAP spot was near the origin and could not be identified. Therefore, we performed back-extraction three times with 50 mL of pure water to remove some of it, and the origin spot (stained with ninhydrin) disappeared. Purification was performed by silica gel column chromatography (Hexane: EtOAc: Acetic acid = 50:50:1, diameter 5 cm, height 10 cm), and compound 4 was obtained as a pale yellow, transparent oil. Identification was 1The product was analyzed by H-NMR (DMSO-d6) and ESI-MS (negative). The yield was 1.724 g, or 64.1%.

[0105] [Compound 5] A 200 mL two-necked recovery flask was charged with AlCl3 (1.523 g, 11.43 mmol, 4.04 eq), dried, and purged with Ar. 30 mL of dry DCM and tetrachlorocyclopropene (0.36 mL, 2.94 mmol, 1.04 eq) were added, in that order, and stirred at room temperature for 10 minutes, then cooled to -20°C. A separate 100 mL recovery flask was charged with compound 4 (0.9989 g, 2.827 mmol, 1 eq), dried, and purged with Ar. 20 mL of dry DCM was added, and the mixture was cooled to -20°C with stirring. The solution containing compound 4 was transferred to the solution containing tetrachlorocyclopropene via cannulation over 10 minutes, and the mixture was stirred at -20°C for 5.5 hours, followed by another 2 hours at room temperature. The disappearance of the raw materials was confirmed by TLC (methanol / dichloromethane=1 / 5, Rf: Compound 4=0.45), and 10 mL of pure water was added and the mixture was vigorously stirred.

[0106] When acetone was added to the oil obtained by concentrating the reaction solution, a gray-white precipitate was formed. The supernatant was removed by decantation, and acetone was added again to wash the residue, a total of three times. The precipitate was then collected by suction filtration to obtain compound 5 as a gray-white solid. 1 Analysis by H-NMR (DMSO-d6, 600 MHz) revealed that the yield was 229 mg, 19.8%.

[0107] [Compound 6] Compound 5 (101.9 mg, 0.2501 mmol, 1 eq), NHS (62.3 mg, 0.5413 mmol, 2.2 eq), and EDC·HCl (142.2 mg, 0.7418 mmol, 3.0 eq) were placed in a 10 mL two-necked eggplant flask, dried, and purged with Ar. 5 mL of dry DCM was added to the mixture, and the mixture was stirred to initiate the reaction. After approximately 18 h, the disappearance of the starting materials was confirmed by TLC (methanol / dichloromethane = 1 / 10, Rf: Compound 5 = 0.11, Compound 6 = 0.42, detected by UV and bromocresol green). The solvent was evaporated, the mixture was suspended in 2 mL of acetone, and the mixture was washed with ultrasound. The precipitate was collected by suction filtration and washed with 8 mL of acetone on a filter paper, yielding compound 6 as a light brown solid. The yield was 86.0 mg, 68%.

[0108] [Compound 7] Compound 6 (39.51 mg, 7.832 × 10-5 mol, 2.52 eq) and HOOC-PEG were placed in a 10 mL two-necked flask. ｎ -NH ２ (MW: 3400, NOF Corporation, product name "Sunbright PA-034HC") (105.81 mg, 3.112 × 10-5 mol, 1 eq) was added and the mixture was dried up and purged with Ar. 3 mL of dry DMF and dry TEA (0.04 mL, 2.9 × 10-4 mol, 9.2 eq) were added and stirred to initiate the reaction. After 40 hours, the disappearance of the starting materials was confirmed by TLC (methanol / dichloromethane = 1 / 5, Rf: PEG = 0.29, compound 7 = 0.32, detected with ninhydrin). The solvent was evaporated and ether precipitation (diethyl ether 40 mL, crude in dichloromethane 1 mL, -15°C, 15,000 G, 10 min) was performed. After air drying, the residue was dissolved in approximately 10 mL of purified water and dialyzed. Dialysis was continued for 31 hours, and then lyophilization yielded compound 7 as a light brown solid. Identification was confirmed. 1 H-NMR (DMSO-d6) and MALDI-TOF-MS (Dithranol & NaCl, positive) showed that the yield was 100.8 mg, or 85%.

[0109] [Compound 8] Compound 7 (19.58 mg, 5.17°10-6 mol) was placed in a centrifugal tube, dissolved in 1 mL of DMSO-d6, and irradiated with 8 J of light using a light irradiator. 1 When the reaction was confirmed by H-NMR (DMSO-d6), a small amount of raw material was observed, so a UV light was applied to the reaction mixture until the raw material disappeared. 1 Identification was carried out by H-NMR (DMSO-d), and the mixture was added with pure water and lyophilized to obtain Compound 8 as a light brown solid. The yield was 19.0 mg, 97%.

[0110] [Compound 1] NHS (4.06 mg, 3.53 × 10 mol, 6.4 eq) and DCC (12.75 mg, 6.65 × 10 mol, 12 eq) were placed in a 10 mL two-necked recovery flask, dried, and purged with Ar. Compound 8 (19.0 mg, 5.01 × 10 mol, 1 eq) dissolved in 3 mL of dry DCM was added and stirred to initiate the reaction. After approximately 26 h, completion of the reaction was confirmed by TLC (methanol / dichloromethane = 1 / 5, Rf: Compound 8 = 0.58). The solvent was evaporated and the residue was redissolved in approximately 1 mL of DCM. Ether precipitation (-15°C, 15,000 G, 10 min) was performed with 40 mL of diethyl ether. The supernatant was removed and air-dried. 3 mL of DCM was added and filtered through a cotton plug. The solvent was evaporated to obtain Compound 1 as a light brown solid. The yield excluding the remaining DCC urea was 12.8 mg, a 66% yield. 1 H-NMR (DMSO-d6) was performed.

[0111] <Synthesis of Compound 2> Similarly, Compound 2 (pDBCO-PEG-NHS) having a photoactivatable dibenzocyclooctyne was synthesized according to the following synthesis scheme. The synthesis of Compounds 3 to 7 was the same as that of Compound 1 above.

[0112] [Compound 2] Compound 7 (20.99 mg, 5.54 × 10 mol, 1 eq), NHS (4.06 mg, 3.53 × 10 mol, 6.4 eq), and DCC (12.75 mg, 6.65 × 10 mol, 12 eq) were placed in a 50 mL two-necked flask, dried, and purged with Ar. 3 mL of dry DCM was added and stirred to initiate the reaction. After approximately 26 h, completion of the reaction was confirmed by TLC (MeOH / DCM = 1 / 5, Rf: Compound 7 = 0.45, Compound 2 = 0.58). The solvent (a cotton plug filter was omitted) was evaporated, and the residue was redissolved in approximately 1 mL of DCM and subjected to ether precipitation with 40 mL of diethyl ether (-15°C, 15,000 G, 10 min). The supernatant was removed and air-dried, and 3 mL of DCM was added and filtered through a cotton plug. The solvent was distilled off to obtain Compound 2 as a light brown solid. The yield excluding the remaining DCC urea was 20.8 mg, and the yield was 96.6%. 1 H-NMR (DMSO-d6) and MALDI-TOF MS (positive, matrix: dithranol and NaCl) were used.

[0113] Example 2: Synthesis of a surface modifier having a cell attachment site (second surface modifier) ​​Next, a second surface modifier was synthesized by introducing an azide group into a cell adhesion molecule via a photodegradable linker. Specifically, two types of surface modifiers were synthesized: Compound a (cRGD-PL-Azide), in which an azide group was introduced via a photodegradable linker into the amino group of the lysine side chain of a cyclized RGD peptide (cRGD: Funakoshi, Cyclo(-RGDfK)); and Compound b (Lipid-PEG-PL-Azide), in which an azide group was introduced via a photodegradable linker into the terminus of PEG-lipid.

[0114]

[0115] In the chemical formula on the left side, compound a is compound where "R" is a cyclized RGD peptide, and compound b is compound where "R" is a PEG lipid. When these compounds are irradiated with UV light, the 2-nitrobenzyl moiety, which is a photolabile linker, is cleaved. This allows the recovery of microcytes immobilized on the cell adhesion molecules.

[0116] Example 3: Modification and Evaluation of Block Layer <Block Layer Formation> A glass slide was immersed in an alkaline solution (approximately 5% Contaminon aqueous solution) for 18 hours, followed by immersion in pure water, acetone, isopropanol, and acetone, successively, for 10 minutes each while applying ultrasonic waves, to clean the substrate surface. The slide was then immersed in a 1% APTES solution (ethanol solution) at room temperature for 30 minutes to introduce amino groups. The surface was then rinsed with methanol and then cleaned in methanol for 10 minutes while applying ultrasonic waves. A 4-branched PEG having an N-hydroxysuccinimide (NHS) ester at its terminus (4arm-PEG-NHS: molecular weight approximately 10,000, manufactured by NOF Corporation, Sunbright PTE100GS) and a 4-branched PEG having an amino group at its terminus (4arm-PEG-Amine: molecular weight approximately 10,000, manufactured by NOF Corporation, Sunbright PTE100PA) were dissolved in ethanol to final concentrations of 2.5, 5.0, and 10 μM, respectively. First, 90 μL of the 4arm-PEG-NHS solution was cast onto the amino-functionalized glass slide and allowed to air dry. Next, the 4arm-PEG-Amine solution was cast onto the glass slide and allowed to air dry. This procedure was repeated 2 to 8 times (Figure 1).

[0117] Evaluation of the Blocking Layer: A microchamber (ibidi, width: 3.8 mm, length: 18 mm, height: 0.40 mm) was attached to the surface, and the channel was washed five times with 1 mL of ultrapure water. Then, phosphate buffered saline (PBS) was introduced into the channel to replace the internal surface. Human cervical cancer cells (HeLa cells) were detached by trypsinization and suspended in serum-containing medium (DMEM medium, containing 10% bovine serum albumin (FBS)) (5 ​​× 105 cells / mL) before being introduced into the microchannel. After 22 hours of incubation in an incubator at 37°C and 5% CO2, the channel was washed three times with PBS. The cells attached to the bottom of the channel were observed under a microscope. Figure 2 shows images of the substrate surface before and after seeding with HeLa cells. Figure 3 is a graph showing the number of stacked blocking layers and the specific area occupied by cells.

[0118] As a result, cell adhesion was no longer observed when the layer was stacked six times or more at 2.5 μM, four times or more at 5.0 μM, and two times or more at 10 μM. This confirmed that cell adhesion could be almost completely suppressed by providing a layer of 4-arm PEG alternatingly stacked blocking layers on the substrate surface.

[0119] Example 4: Preparation of surface-modified substrate A substrate on which a blocking layer had been formed was modified with the surface modifiers synthesized in Examples 1 and 2 to prepare a surface-modified substrate.

[0120] As in Example 3, a 100 μM 4arm-PEG-NHS solution and a 4arm-PEG-Amine solution were cast sequentially onto an APTES-loaded glass slide, forming a 4arm-PEG block layer. Next, a 20 μM solution of compound 2 (pDBCO-PEG-NHS), a photoactivatable dibenzocyclooctyne (DBCO) precursor, was dissolved in ethanol as the first surface modifier. This solution was then cast onto the glass slide and air-dried. After photoirradiation, the pDBCO-PEG-NHS-modified surface binds to molecules bearing azide groups (Kosaka T., et al., J. Am. Chem. Soc. 2022, 144, 17980-17988).

[0121] Next, a mixed aqueous solution containing 100 μM compound a (cRGD-PL-Azide) and 1 μM compound b (Lipid-PEG-PL-Azide) was prepared as a second surface modifier. A microchamber was attached to the pDBCO-PEG-NHS-modified glass slide, and various patterns of light (wavelength: 365 nm) were irradiated using a maskless exposure system (NeoArc, PALET). The mixed aqueous solution was then introduced into the channel and allowed to stand at room temperature for 10–60 minutes. The mixed aqueous solution was then removed from the channel and washed five times with PBS. This resulted in two cell adhesion molecules being modified via photocleavable linkers only in the light-irradiated area. Here, PEG-lipid is a molecule that rapidly (within minutes) allows cells to adhere to the desired substrate surface, while cRGD is a molecule that allows adherent cells to adhere and spread over a long period of time via membrane proteins such as integrins.

[0122] Example 4: Immobilization of Adherent Cells HeLa cells (5 × 10 cells / ml) suspended in serum-free DMEM medium were introduced into the surface-modified substrate (microchannel) prepared in Example 3. After leaving the substrate to stand for 10 minutes, the medium was washed three times with PBS. The medium was replaced with serum-containing DMEM, the channel was wrapped in aluminum foil, and the medium was cultured in an incubator for several days. The cells were removed from the incubator every day and observed under a microscope. The resulting images are shown in Figure 4.

[0123] The top image in Figure 4 shows microscopic images (images taken after 0, 1, and 2 days of culture) of cells placed on a collagen-coated substrate whose surface was modified with photoreactive molecules; the middle image shows a similar image on a substrate whose APTES surface was modified with photoreactive molecules and linear PEG; and the bottom image shows a similar image on a substrate whose APTES surface was modified with photoreactive molecules after a blocking layer was laminated on it.

[0124] As a result, on the surface-modified substrate of the present invention, cells adhered only to the light-irradiated areas and continued to adhere and spread without leaving the light-irradiated areas even after two days of culture (Figure 4, bottom). On the other hand, on a comparative example, on a collagen surface directly modified with pDBCO-PEG-NHS, cells adhered only to the light-irradiated areas immediately after seeding, but after one day of culture, they spread and migrated to the non-irradiated areas (Figure 4, top). Furthermore, when the APTES surface was directly modified with pDBCO-PEG-NHS and the remaining amino groups were then modified with linear PEG with a molecular weight of 2000 to suppress non-specific cell adhesion and spreading, cells still spread and migrated to the non-irradiated areas after one day of culture (Figure 4, middle). Thus, by suppressing non-specific cell adhesion and spreading by the addition of a blocking layer, adherent cells were able to remain in the desired area even after culture after light irradiation.

[0125] Example 6: Recovery of spheroids Next, the cells immobilized and cultured in Example 4 were recovered. Figure 5A shows a conceptual diagram of the process of detaching cells by light irradiation.

[0126] Cells were arranged in various shapes using the same procedure as in Example 4 and cultured for 5 days. When a portion of the rod-shaped spheroids prepared in large quantities on a substrate was irradiated with light (wavelength: 365 nm, light intensity: 10 J / cm) using a mercury lamp, the spheroids detached from the substrate surface and were confirmed to flow out of the channel by washing (Figure 5B). Figure 4B shows a microscopic image of the rod-shaped spheroid array after generation (left), a microscopic image of the spheroids after partial irradiation with light to detach them (center), and a microscopic image of the spheroids after washing and recovery of the irradiated region (right).

[0127] Furthermore, we attempted to recover sheet-like spheroids by adjusting the irradiation area. Specifically, we irradiated the rectangular area (length: 600 μm × width: 500 μm, aspect ratio: 1.2) shown in Figure 5C with light, placed HeLa cells, and cultured them. Afterwards, we disassembled the microchannel and irradiated the cells with light in the same manner as above. The procedure is outlined in Figure 4D.

[0128] The detached sheet-like spheroids were collected using a pipette and transferred to a culture dish for microscopic observation. After 5 days, the spheroids observed on the substrate (length: 530 μm x width: 400 μm, aspect ratio: 1.3) had shrunk slightly, but still retained the desired shape (length: 460 μm x width: 350 μm, aspect ratio: 1.3) (Figure 5E). Figure 5E shows a microscopic image of the sheet-like spheroid array after generation (left), and microscopic images of the detached spheroids collected by light irradiation and transferred to a culture dish, observed from above (center) and from the side (right). These results confirmed that spheroids with the desired uniform shape could be prepared without damaging intercellular adhesion and causing shape collapse.

Claims

1. A surface-modified substrate for culturing and recovering adherent cells, comprising: a block layer on an upper portion of a substrate that does not have adhesiveness to the adherent cells; a first modified layer modified with a first surface modifying agent on an upper portion of the block layer; and a second modified layer modified with a second surface modifying agent on an upper portion of the first modified layer, the first surface modifying agent comprising a compound having the structure shown below: L-M-N (wherein L is a bonding moiety that bonds to the surface of the block layer; M is a hydrophilic linker; and N is a linking group that covalently bonds to an azide group or a thiol group); the second surface modifying agent comprising a compound having the structure shown below: X-Y-Z (wherein X is a hydrophilic chain having an azide group or a thiol group at an end; Y is a photodegradable linker that is cleaved by irradiation with light; and Z is a cell attachment site that binds to the adherent cells through interaction).

2. The surface-modified substrate according to claim 1, having a patterned surface modification in which the second modification layer is present only on predetermined surface regions of the first surface modifier.

3. The surface-modified substrate according to claim 1, wherein the block layer has a laminated structure of 4-branched or 8-branched polyalkylene glycol.

4. The surface modifier of claim 1, wherein the second surface modifier is a mixture of a compound having a cell-binding polypeptide as the cell attachment site Z and a compound having a hydrophobic chain as the cell attachment site Z.

5. The surface modifier according to claim 4, wherein the molar ratio of the compound having a cell-binding polypeptide to the compound having a hydrophobic chain is in the range of 1000:1 to 1:

1.

6. The surface-modified substrate according to claim 1, wherein the linking group N comprises an alkynyl group or an alkynyl group to which a protecting group has been introduced.

7. The surface-modified substrate according to claim 1, wherein the linking group N has a structure including dibenzocyclooctyne (DBCO) or a precursor thereof.

8. The surface-modified substrate according to claim 1, wherein the substrate-binding moiety L has a substituent capable of binding to the surface of the blocking layer by a covalent bond.

9. The surface-modified substrate according to claim 1, wherein the adherent cells after the culture are a cell mass.

10. A surface-modified substrate for culturing and recovering adherent cells, comprising: a block layer on an upper portion of the substrate that is not adhesive to the adherent cells; and a modification layer on an upper portion of the block layer that has a region to which the adherent cells selectively bind; the modification layer is formed from a surface modifying agent that contains a compound having a cell-binding polypeptide and a compound having a hydrophobic chain, and the molar ratio of the compound having the cell-binding polypeptide to the compound having a hydrophobic chain is in the range of 1000:1 to 1:

1.

11. A surface-modified substrate for culturing and recovering adherent cells, comprising: a block layer on an upper portion of the substrate that has no adhesiveness to the adherent cells; and a modification layer on an upper portion of the block layer that has a region to which the adherent cells selectively bind, wherein the block layer has a laminated structure in which 4-branched or 8-branched polyalkylene glycol is crosslinked.

12. A method for culturing and recovering adherent cells, comprising the steps of: bringing a solution containing predetermined target cells into contact with a surface-modified substrate according to any one of claims 1 to 11, and immobilizing the target cells on the surface of the surface-modified substrate; culturing the immobilized target cells; and irradiating the surface-modified substrate with light to cleave a photodegradable linker present in the modification layer of the surface-modified substrate, thereby separating and recovering the immobilized target cells from the surface-modified substrate.

13. The method according to claim 12, wherein the separated and recovered target cells form a cell mass.

14. A method for producing a surface-modified substrate for culturing and recovering adherent cells, comprising: (A) forming a block layer on an upper portion of a substrate, the block layer having no adhesiveness to the adherent cells; (B) adding a first surface modifier onto the block layer to form a first modified layer, the first surface modifier comprising a compound having the following structure: L-M-N (wherein L is a bonding portion that bonds to the surface of the block layer, M is a hydrophilic linker, and N is a linking group that covalently bonds to an azide group or a thiol group); (C) adding a second surface modifier onto the first modified layer, the second surface modifier comprising a compound having the following structure: X-Y-Z (wherein X is a hydrophilic chain having an azide group or a thiol group at its terminal, Y is a photodegradable linker that is cleaved by irradiation with light, and Z is a cell attachment site that binds to the adherent cells through interaction); (D) forming a second modified layer on the first modified layer by forming a covalent bond between the linking group N in the first surface modifier and an azide group or a thiol group at the end of the hydrophilic chain X in the second surface modifier.

15. The method according to claim 14, further comprising, prior to step (C), a step of irradiating a specific wavelength of light onto a predetermined area on the surface of the first modified layer, thereby forming a patterned surface modification in which the second surface modifier is present only in the predetermined area.

16. The method of claim 14, wherein the second surface modifier is a mixture of a compound having a cell-binding polypeptide as the cell attachment site Z and a compound having a hydrophobic chain as the cell attachment site Z, and the molar ratio of the compound having the cell-binding polypeptide to the compound having the hydrophobic chain is in the range of 1000:1 to 1:1.

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