JP2023165489A - Method for manufacturing substrate for cell immobilization - Google Patents

Abstract

To provide a method for manufacturing a substrate for cell immobilization that enables immobilization and separation of target cells such as iPS cells without damaging the cells and without requiring addition of additives.SOLUTION: A method for manufacturing a substrate for cell immobilization for immobilizing a predetermined target cell to a substrate is provided, including the steps of: (A) modifying the entire or a part of the surface of the substrate with a first surface modification agent including a compound with structure represented by the formula: L-M-N (where L is a substrate binding part, capable of binding to the substrate surface, M is a hydrophilic chain, and N is a linking group, capable of covalently bonding with an azido group); (B) adding, to the substrate, a second surface modification agent that includes a cell-binding protein capable of selectively binding to target cells, with an azido group linked to the cell-binding protein; and (C) forming a covalent bond between a linking group N in the first surface modification agent and the azido group in the second surface modification agent, to obtain a substrate for cell immobilization in which the substrate surface has been modified with the cell-binding protein.SELECTED DRAWING: None

Description

The present invention relates to a method for manufacturing a cell immobilization substrate for selectively immobilizing target cells such as stem cells, and a method for immobilizing and recovering cells using the cell immobilization substrate.

Human induced pluripotent stem cells (iPS) cells have both pluripotency, which allows them to differentiate into all types of cells, and self-renewal ability, which allows them to proliferate indefinitely. Since iPS cells can be prepared from various somatic cells by simply introducing four types of genes, they are expected to be applied to cell therapy and regenerative medicine, which replenish and treat cells and tissues that have lost their functions due to disease, and are currently being used in practice. Clinical application is underway (Non-Patent Document 1). In addition, iPS cells are prepared mainly from cells of patients with congenital diseases, and pathological models for basic research and drug discovery are being developed (Non-Patent Document 2). Although recent technological developments have made the induction efficiency of iPS cells considerably higher, depending on the origin and cell line of the original cells, the percentage of cells that have acquired high undifferentiated potential may be extremely low. many. Therefore, a technique for selectively isolating iPS cells from a cell population is an important technique that is key to the application of iPS cells.

On the other hand, in medical applications, undifferentiated iPS cells or insufficiently differentiated cell types are mixed into therapeutic cells and tissues prepared by differentiating iPS cells, leading to canceration and tissue malformation. It has become a problem to cause this (Non-Patent Document 3). Therefore, a technique for selectively removing iPS cells from therapeutic cells prepared by differentiation from iPS cells is also essential.

Additionally, if it is possible to capture iPS cells only in a specific region of the substrate, it is possible to clarify the influence of cell patterns on differentiation, and to induce differentiation after patterning cells that are difficult to pattern in advance. , can be a useful tool for basic research and tissue engineering. In recent years, the diversity of individual cells has attracted attention, and it is known that there are large variations in the differentiation ability of iPS cells in particular. Therefore, if it is possible to construct a single-cell array of iPS cells and comprehensively observe variations in their differentiation process, it will lead to the development of a technology that predicts the differentiation potential of iPS cells from information such as their initial morphology. Therefore, a technique to capture iPS cells only in a desired area is also extremely important.

In this regard, classical techniques for isolating iPS cells include colony picking and selection methods in which foreign antibiotic resistance genes are expressed by transcription factors specific to pluripotent cells (such as “Nanog”). has been used (Non-patent Document 4). However, the former method has the problem of poor accuracy, and the latter method may hinder its application to therapy due to the introduction of unnecessary foreign genes. A similar problem exists in the technology of expressing fluorescent protein genes and selecting them by FACS. A technique has been reported to isolate iPS cells by FACS or MACS using antibodies against cell surface molecules specific to undifferentiated cells (undifferentiated markers, TRA-1-60, SSEA-4) (Non-patent Document 5). and 6). However, it has been pointed out that FACS physically damages cells in the first place, and magnetic beads accumulated on the cell surface or inside cells in MACS pose a problem in subsequent medical applications. Therefore, there is a need for a technique for isolating iPS cells without damaging the cells and without adding genes or additives that would cause problems in subsequent applications.

On the other hand, it is also possible to use FACS as a technique for removing iPS cells contaminating therapeutic cells, but this poses the problem of physical damage to the cells. A method has been reported to utilize the difference in energy metabolism between iPS cells and differentiated cardiomyocytes to remove iPS cells by culturing them in a lactose-based medium that kills cells other than cardiomyocytes (non-patent literature). 7) However, such a technique can only be applied to cardiomyocytes. In contrast, by synthesizing messenger RNA that switches the expression of antibiotic resistance genes by sensing microRNA (miRNA-302), which is highly active only in pluripotent stem cells, and introducing it into the cells, iPS cells A method has been developed to specifically identify and remove only partially differentiated iPS cells by reducing their antibiotic resistance (Non-Patent Document 8). However, the introduction of a foreign antibiotic resistance gene into therapeutic cells as in this method poses a problem for subsequent medical applications. Therefore, there is also a need for a technique to remove iPS cells without damaging the cells or adding genes.

Mandai, M., et al., N. Engl. J. Med. 2017, 376, 1038-1046 Matsumoto, R., et al., J Clin Invest. 2020, 130, 641-6540 Koyanagi-Aoi, M., et al., Natl. Acad. Sci. USA 2013, 110, 20569-20574 Hotta A., et al., Nat. Methods 2009, 6, 370-376 Valamehr B., et al., Sci. Rep. 2012, 2, 00213 Yang W., et al., PLoS ONE 2015, 10, e0134995 Tohyama H., et al., Cell Stem Cell 2013, 12, 127-137 Parr C. J. C., et al., Sci. Rep. 2016, 6, 32532

Therefore, in order to solve the problems of conventional iPS cell isolation and removal methods, the present invention aims to produce iPS cells without damaging the cells or adding additives that would pose problems for subsequent medical applications. The objective of this project is to develop a new method that can immobilize and sort target cells such as

As a result of intensive studies to solve the above problems, the present inventors have discovered that the binding activity of cell-binding proteins that can selectively capture only target cells such as iPS cells with sufficient strength is impaired. The present invention was completed by developing a technology that can modify the surface of a base material without any modification.

That is, in one aspect, the present invention relates to a method for producing a cell immobilization substrate with surface modification capable of selectively capturing target cells such as iPS cells, and more specifically,
<1> A method for producing a cell immobilization substrate for immobilizing predetermined target cells on the substrate, the method comprising: (A) modifying the entire or part of the surface of the substrate with a first surface modifier; a step in which the first surface modifier includes a compound having the structure shown below,
L-M-N
In the formula, L is a substrate binding part that can be bonded to the surface of the substrate, M is a hydrophilic chain, and N is a linking group that can be covalently bonded to an azide group; B) adding onto the substrate a second surface modifier that includes a cell-binding protein that can selectively bind to the target cells and has a structure in which an azide group is linked to the cell-binding protein; (C) The cell-binding protein was modified on the surface of the base material by forming a covalent bond between the linking group N in the first surface modifier and the azide group in the second surface modifier. The manufacturing method includes a step of obtaining a cell immobilization substrate;
<2> The manufacturing method according to <1> above, including a step of irradiating the surface of the base material modified with the first surface modifier with light of a specific wavelength before the step (B);
<3> The manufacturing method according to <1> above, wherein the cell immobilization substrate has a patterned surface modification in which the cell-binding protein is present only in a specific surface region;
<4> The production method according to <1> above, wherein the second surface modifier has 1 to 2.5 azide groups per molecule of the cell-binding protein;
<5> The manufacturing method according to <1> above, wherein the connecting group N includes an alkynyl group or a group in which a protecting group is introduced into an alkynyl group;
<6> The manufacturing method according to <1> above, wherein the linking group N has a structure containing dibenzocyclooctyne (DBCO) or a precursor thereof;
<7> The manufacturing method according to <1> above, wherein the hydrophilic chain M includes a hydrophilic polymer;
<8> The manufacturing method according to <1> above, wherein the hydrophilic chain M contains polyalkylene glycol;
<9> The manufacturing method according to <1> above, wherein the base material bonding portion L has a substituent that can be bonded to the surface of the base material through a covalent bond;
<10> The manufacturing method according to <1> above, wherein the target cells are stem cells;
<11> The manufacturing method according to <10> above, wherein the stem cells are induced pluripotent stem cells (iPS cells) or embryonic stem cells (ES cells);
<12> The production method according to <1> above, wherein the cell-binding protein is a lectin or an antibody;
<13> The production method according to <12> above, wherein the lectin is BC2LCN lectin or a variant thereof;
It provides:

In another aspect, the present invention also relates to a cell immobilization method and a cell recovery method using the above cell immobilization substrate, and more specifically,
<14> A method of immobilizing cells on a cell immobilization substrate obtained by the production method according to any one of <1> to <13> above, the method comprising: The method includes the step of immobilizing the target cells on the surface of the cell immobilization substrate by contacting the target cells with a solution containing the target cells;
<15> A method for recovering cells immobilized by the method described in <14> above, wherein the surface of the cell immobilization substrate is treated with a sugar solution or an enzyme solution. The method includes the step of separating and collecting the target cells from the cell immobilization substrate.
It provides:

＜１９＞細胞固定化用基材の表面修飾に用いるためのキットであって上記＜１６＞～＜１８＞のいずれか１に記載の表面修飾剤、及び、標的細胞と選択的に結合し得る細胞結合性タンパク質にアジド基を連結した構造を有する化合物を格納したキット；
＜２０＞前記細胞結合性タンパク質が、レクチン又は抗体である、上記＜１９＞に記載のキット；
＜２１＞細胞固定化用基材であって、
以下の構造：
Ｌ－Ｍ―Ｎ
（式中、Ｌは、基材の表面に結合し得る基材結合部であり、Ｍは、親水性鎖であり、Ｎは、アジド基と共有結合し得る連結基である。）。
で表される化合物を含む、第１の表面修飾剤；及び、所定の標的細胞と選択的に結合し得る細胞結合性タンパク質を含み、当該細胞結合性タンパク質にアジド基を連結した構造を有する第２の表面修飾剤で修飾された表面を有する、該基材
を提供するものである。 In a further aspect, the present invention provides a surface-modifying agent for a substrate suitable for surface modification of the above-mentioned substrate for cell immobilization, a kit containing the surface-modifying agent, and a surface-modifying agent for cell immobilization modified with the surface-modifying agent. Regarding the base material, more specifically,
<16> A surface modifying agent for a substrate for immobilizing predetermined target cells,
Structure below:
L-M-N
(In the formula, L is a substrate binding part that can be bonded to the surface of the substrate, M is a hydrophilic chain, and N is a linking group that can be covalently bonded to an azide group.) The surface modifier comprises a compound represented by;
<17> The surface modifier according to <16> above, wherein the linking group N has a structure containing dibenzocyclooctyne (DBCO) or a precursor thereof;
<18> The surface modifier according to <16> above, selected from compounds having the following structures:

<19> A kit for use in surface modification of a substrate for cell immobilization, which is capable of selectively binding to the surface modifying agent according to any one of <16> to <18> above and target cells. A kit containing a compound having a structure in which an azide group is linked to a cell-binding protein;
<20> The kit according to <19> above, wherein the cell-binding protein is a lectin or an antibody;
<21> A base material for cell immobilization,
Structure below:
L-M-N
(In the formula, L is a substrate binding part that can be bonded to the surface of the substrate, M is a hydrophilic chain, and N is a linking group that can be covalently bonded to an azide group.)
A first surface modifying agent comprising a compound represented by The present invention provides the base material having a surface modified with the surface modifier of No. 2.

According to the present invention, the following effects that could not be achieved with conventional techniques can be achieved:
(1) The surface of the substrate can be modified while maintaining the binding activity of cell-binding proteins such as lectins, which allows target cells such as iPS cells to be selectively captured at high density:
(2) Only the target cells can be immobilized on the surface of the substrate by an extremely simple and quick operation of seeding the target cells on the cell immobilization substrate and removing uncaptured free cells by washing etc.:
(3) Immobilized target cells can be easily recovered using operations similar to conventional general methods:
(4) By using a photoresponsive surface modifier, target cells can be arranged and immobilized in precise patterns.
(5) The cell immobilization substrate of the present invention modified with a surface modifier can be stored in a dry state for a long period of time.

For example, by applying the present invention to the immobilization and recovery of iPS cells, it is expected that it will be put to practical use as a tool for easily purifying high-quality iPS cells. Conventionally, variations in the quality of iPS cells and contamination of iPS cells with therapeutic cells have been major obstacles to clinical application, but the present invention enables application to the cell processing industry using iPS cells as a cell resource. This can be an extremely inexpensive and simple solution to these challenges.

FIG. 1 shows the amount of azide group modification of rBC2LCN by gel shift assay for the lectin-containing surface modifier synthesized in Examples. Figure 2 (a) is a fluorescence image of Calcein-AM stained iPS cells selectively captured in the light irradiation area on a surface modified with a DBCO-containing surface modifier (compound 2); This is a graph quantifying the cell density on the surface of a substrate using the method (UV+: with light irradiation, UV-: without light irradiation). FIG. 3 is a schematic diagram showing the procedure of the iPS cell capture experiment of Example 3. Figure 4 shows the case of using a low-azide lectin (rBC2LCN-azide with an average modification amount of 1.1 azide groups) and a high-azide lectin (rBC2LCN-azide with an average modification amount of 3.0 azide groups). , are fluorescent images showing captured Calcein-AM stained iPS cells. Figure 5a is a fluorescence microscopy image of cells immobilized when a mixed suspension containing two types of cells was applied to a substrate modified with a lectin-containing surface modifier (rBC2LCN-azide); Figure 5b This is a fluorescence microscopy image of cells immobilized when a mixed suspension containing two types of cells was applied to a substrate modified with azidated PEG lipid (Oleyl-PEG-azide) that does not have lectin. It is. Figure 5c is the molecular structure of Oleyl-PEG-azide. Figure 5d is a fluorescence microscopy image of cells captured on the substrate surface modified with rBC2LCN-azide after treatment with protease; Figure 5e is a fluorescence microscopy image of cells captured on the substrate surface modified with rBC2LCN-azide; Figure 3 is a fluorescence microscopy image of cells on a substrate after treatment of cells captured on the surface with proteolytic enzymes. FIGS. 5f and 5g are graphs obtained by quantifying changes in cell density before and after enzyme treatment on substrates modified with rBC2LCN-azide and Oleyl-PEG-azide using image analysis software. Figure 6a is a fluorescence microscopy image of cells captured when a mixed suspension of iPS cells and iPS cell-derived neural progenitor cells was applied to a substrate modified with a lectin-containing surface modifier (rBC2LCN-azide). Yes; Figure 6b shows the results when a mixed suspension of iPS cells and iPS cell-derived neural progenitor cells was applied to a substrate modified with azidated PEG lipid (Oleyl-PEG-azide) that does not have lectin. This is a fluorescence microscopy image of captured cells (neural progenitor cells are stained with red fluorescent dye and undifferentiated iPS cells are stained with rBC2LCN-FITC). FIG. 6c is a graph quantified by image analysis of the cell density in the light irradiated area on the substrate modified with rBC2LCN-azide and Oleyl-PEG-azide.

Embodiments of the present invention will be described below. The scope of the present invention is not limited to these explanations, and other than the examples below may be modified and implemented as appropriate without departing from the spirit of the present invention.

1. Method for manufacturing a substrate for cell immobilization of the present invention A first aspect of the present invention is a method for manufacturing a substrate for cell immobilization for immobilizing predetermined target cells on a substrate, comprising the following steps. Characterized by including (A) to (C):
(A) a step of modifying the whole or part of the surface of the base material with a first surface modifier, the first surface modifier including a compound having the structure shown below,
L-M-N
In the formula, L is a substrate binding part that can be bonded to the surface of the substrate, M is a hydrophilic chain, and N is a linking group that can be covalently bonded to an azide group;
(B) adding onto the substrate a second surface modifier that includes a cell-binding protein that can selectively bind to the target cell and has a structure in which an azide group is linked to the cell-binding protein; and (C) modifying the cell-binding protein on the surface of the base material by forming a covalent bond between the linking group N in the first surface modifying agent and the azide group in the second surface modifying agent. A step of obtaining a substrate for cell immobilization.

That is, first, in step (A), a first surface modifier is added to modify the surface of the base material, and then, in step (B), a second surface modifier is further added onto the base material. Surface modification of the base material is performed in two steps. Here, since the first surface modifier has a linking group that can covalently bond with an azide group in its molecule, when the second surface modifier is added, the azide group that the second surface modifier has in its molecule By reacting with the group, in step (C), a second surface modifier is linked to the upper layer of the first surface modifier by a covalent bond, and a cell immobilization agent having the second surface modifier on the outermost surface is formed. A base material can be obtained. Since the second surface modification agent has a cell-binding protein that can selectively bind to target cells, the target cells are selectively captured by the cell-binding protein modified on the substrate surface. and can be immobilized on the surface of the substrate.

Target cells to be immobilized can include animal cells, plant cells, insect cells, prokaryotic cells, fungal cells, etc., and generally do not adhere or spread on the surface of a carrier such as a culture device, but are suspended or precipitated. So-called "floating cells" (e.g. blood cells) that proliferate under certain conditions and "adherent cells" that adhere and spread on the carrier surface are dispersed from the carrier using an appropriate dispersant such as EDTA-trypsin or dispase, and temporarily dispersed. This includes suspended cells (for example, fibroblasts detached from the carrier with an EDTA solution) and cells adhered to the carrier. It also includes living organisms that have a phospholipid double membrane on the surface, such as liposomes, exosomes, bacteria, viruses, organelles, and plant cells with cell walls removed (protoplasts). Moreover, according to the base material for cell immobilization of the present invention, in addition to these, substances having lipids such as lipid-coated particles can also be immobilized.

Preferably the target cells are stem cells. "Stem cells" are generally defined as undifferentiated cells that have "self-renewal ability" that allows them to proliferate while maintaining an undifferentiated state, and "pluripotency" that allows them to differentiate into all three germ layer lineages. Stem cells (pluripotent stem cells) to be immobilized using the cell immobilization substrate of the present invention are undifferentiated cells having self-renewal ability and pluripotency, and are at least pluripotent stem cells. cells) and multipotent stem cells. Stem cells include, in particular, pluripotent stem cells such as embryonic stem cells (ES cells) and induced pluripotent cells (iPS cells) obtained by introducing reprogramming factors into somatic cells. can be mentioned. Note that multipotent stem cells include somatic stem cells such as mesenchymal stem cells, hematopoietic stem cells, nervous system stem cells, bone marrow stem cells, and germinal stem cells. In the present invention, the term "cell" is used to include both stem cells and differentiated cells (somatic cells). In the present invention, target cells are preferably iPS cells. Therefore, the cell immobilization substrate of the present invention is particularly suitable for immobilizing iPS cells.

Each step in the manufacturing method of the present invention will be specifically explained below.

1-1. Process (A)
As mentioned above, step (A) is a step of modifying the entire or part of the surface of the base material with the first surface modifier, whereby a layer of the first surface modifier is formed on the base material. be done. The first surface modifier layer can be said to be a so-called base layer to which the second surface modifier is connected in a later step. The first surface modifier is preferably arranged in the form of a monolayer on the surface of the base material.

<First surface modifier>
The first surface modifier has three sites: a site for binding to the surface of the base material, a site for a hydrophilic chain, and a site for linking with the azide group in the second surface modifier. Specifically, the first surface modifier includes a compound having the structure shown below.
L-M-N

In the formula, L is a substrate binding part that can be bonded to the surface of the substrate, M is a hydrophilic chain, and N is a linking group that can be covalently bonded to an azide group.

The base material binding portion L preferably has a substituent that can be bonded to the surface of the base material through 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. As described later, when using a coating layer such as collagen on the surface of the base material, a functional group that can bond with the coating layer can be used. For example, in the case of a collagen coating layer, an amino group in collagen can be used. An active ester group that can be covalently bonded to is preferred, and it is particularly preferred to have an NHS group.

For example, the base material bonding portion L can use the functional group shown below (in the formula, the arrow represents the bonding point to the hydrophilic chain M).

Note that it is also possible to immobilize the first modifier on the surface of the substrate by employing a polypeptide or a nucleic acid as the substrate binding portion L and reacting with a substrate that has a substance on the surface that can bind to the polypeptide or nucleic acid. For example, a combination of complementary DNA strands; a combination of biotin and avidin, etc. can also be used.

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

As the linking group N, any functional group known in the art can be used as long as it is a functional group that can covalently bond to the azide group in the second surface modifier. Typically, the linking group N can be an alkynyl group, a group obtained by introducing a protective group into an alkynyl group, or a functional group or partial structure containing these groups. The protecting group in this case is not particularly limited, but includes, for example, cyclic ketones such as cyclopropenone.

The partial structure containing such an alkynyl group or a group in which a protecting group is introduced into an alkynyl group may preferably be a structure containing dibenzocyclooctyne (DBCO) or a precursor thereof. Further, the precursor of DBCO may include a structure in which the alkynyl group portion of DBCO is protected by a protecting group. DBCO may have any substituent.

DBCO may have any substituent. In this specification, when it is defined that "it may have a substituent", the type of substituent, the substitution position, and the number of substituents are not particularly limited, and two or more substituents , 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. Further substituents may be present in these substituents. The substituent in DBCO is not particularly limited, but is preferably an alkoxy group such as a methoxy group.

Non-limiting examples of the linking group N containing DBCO or its precursor include the following structures.

For example, covalent bonds such as an amide bond, an ester bond, an ether bond, a thioether bond, a carbamate bond, a thiocarbamate bond, a triazole bond, and a urea bond can be used to connect the above L, M, and N sites. Note that the connection between the substrate binding portion L and the hydrophilic chain M and the connection between the hydrophilic chain M and the linking group N may be the same and different bonding modes, or may be different bonding modes. can.

Furthermore, in one embodiment, an arbitrary linker can be present between each of the L, M, and N sites. Such a linker is, but is not particularly limited to, for example, a C _6-14 arylene group or a C _1-10 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 with 1 to 5 unsaturated bonds, and the alkylene group Of the carbon atoms in the group, 1 to 4 carbon atoms may be replaced with NH, N (C _1-10 alkyl), O or S. In some cases, it is preferable that between L and M and 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. .

In this specification, "alkyl or alkyl group" may be any aliphatic hydrocarbon group consisting of linear, branched, cyclic, or a combination thereof. _The number _of carbon atoms in the alkyl group is not particularly limited _; ). In this specification, the alkyl group may have one or more arbitrary substituents. For example, C _1-8 alkyl includes methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neo-pentyl, n-hexyl, isohexyl, Includes n-heptyl, n-octyl, etc. Examples of the substituent include an alkoxy group, a halogen atom (which may be a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom), an amino group, a mono- or di-substituted amino group, a substituted silyl group, or Examples include, but are not limited to, acyl. When an alkyl group has two or more substituents, they may be the same or different. The same applies to the alkyl moieties of other substituents (eg, alkoxy groups, arylalkyl groups, etc.) containing alkyl moieties.

As used herein, "alkylene" is a divalent group consisting of a linear or branched saturated hydrocarbon, such as methylene, 1-methylmethylene, 1,1-dimethylmethylene, ethylene, 1-methylethylene, 1-ethylethylene, 1,1-dimethylethylene, 1,2-dimethylethylene, 1,1-diethylethylene, 1,2-diethylethylene, 1-ethyl-2-methylethylene, trimethylene, 1 -Methyltrimethylene, 2-methyltrimethylene, 1,1-dimethyltrimethylene, 1,2-dimethyltrimethylene, 2,2-dimethyltrimethylene, 1-ethyltrimethylene, 2-ethyltrimethylene, 1,1 -diethyltrimethylene, 1,2-diethyltrimethylene, 2,2-diethyltrimethylene, 2-ethyl-2-methyltrimethylene, tetramethylene, 1-methyltetramethylene, 2-methyltetramethylene, 1,1- Examples include dimethyltetramethylene, 1,2-dimethyltetramethylene, 2,2-dimethyltetramethylene, 2,2-di-n-propyltrimethylene and the like.

As used herein, "amide or amide group" refers to both RNR'CO- (alkylaminocarbonyl-, when R = alkyl) and RCONR'- (alkylcarbonylamino-, when R = alkyl). including.

Specific examples of the first surface modifier used in the present invention include Compound 1 and Compound 2 having the following structures. However, it is not limited to these.

<Base material>
The material, shape, etc. of the base material used in the present invention are not particularly limited, and various suitable base materials can be selected depending on the intended use. For example, the shape of the base material to be modified may be substrate-like (plate-like or film-like, e.g. slide glass, dish, microplate, microarray substrate, etc.), carrier (e.g. particle-like such as beads), etc. colloids), fibrous structures, tubes, containers (eg test tubes and vials). Materials for the base material to be modified include: glass; cement; ceramics such as ceramics or fine ceramics; polymer resins such as polyethylene terephthalate, cellulose acetate, polycarbonate, polystyrene, and polymethyl methacrylate; biomaterials such as polypeptides and proteins; silicon; Examples include activated carbon; porous glass; porous ceramics; porous silicon; porous activated carbon; nonwoven fabric; filter paper; membrane filter; and conductive materials such as gold. In order to introduce amino groups, carboxyl groups, hydroxyl groups, etc., the surface of the substrate to be modified can be coated with a polymer such as a polycation, or treated with a silane coupling agent having a substituent introduced onto the surface of the substrate. Alternatively, a reactive functional group may be introduced by plasma treatment.

The first surface modifier may be modified by directly bonding to the surface of the base material, or by providing a coating layer on the surface of the base material and binding the first surface modifier to the surface of the coating layer. Surface modification can also be carried out. As such a coating layer, for example, collagen, bovine serum albumin (BSA), 3-aminopropyltriethoxysilane (APTES), or egg albumin can be used.

Typically, modification of the base material surface with the first surface modifier is performed by bringing a solution containing the first surface modifier into contact with the base material surface. The type of solvent in the solution containing the first surface modifier is not particularly limited, but for example, buffers such as phosphate buffer, borate buffer, Tris buffer, acetate buffer, carbonate buffer, Good's buffer, etc. A solution or an isotonic solution thereof; or an organic solvent such as acetonitrile, dimethyl sulfoxide, dimethylformamide, etc.; or a mixture of the buffer solution or isotonic solution and the organic solvent can be used. In order not to damage or denature the substrate, the first surface modifier is dissolved in an easily soluble organic solvent (for example, acetonitrile, dimethyl sulfoxide, dimethyl formamide), and then the solution is sufficiently diluted with the buffer solution or isotonic solution. may be used. The temperature during contact with the first surface modifier is not particularly limited, but is preferably -78 to 200°C, more preferably 0 to 100°C. The contact time 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 base material with water.

1-2. Process (B)
As described above, step (B) is a step of adding a second surface modifier onto the base material on top of the first surface modifier. The second surface modification agent has in its molecule a cell-binding protein that can selectively bind to target cells, and an azide group linked to the cell-binding protein. The cell-binding protein is a site for capturing target cells and immobilized on the modified surface of the base material, and the azide group is a site for binding to the linking group N in the first surface modifier. be.

As the cell-binding protein, a known protein that can bind to the target cell can be used depending on the type of target cell to be immobilized. Typical examples of such cell-binding proteins include lectins and antibodies. Other examples of proteins that bind to cell surfaces include cell adhesion proteins such as collagen, fibronectin, vitronectin, and laminin; and proteins involved in tight junctions such as E-cadherin. Further, the cell-binding protein in the present specification may include a polypeptide molecule in a broad sense, for example, a peptide having a partial structure of a cell adhesion protein, such as an RGD peptide or a polyarginine peptide, or a cationic cell-penetrating peptide.

As used herein, "lectin" refers to the partial structure or overall structure of sugar chains bound to complex carbohydrates such as glycoproteins, glycolipids, proteoglycans, glycopeptides, lipopolysaccharides, peptidoglycans, and glycosides such as steroid compounds. , or a protein that recognizes a glycopeptide moiety 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, viral hemagglutinins, and the like. Lectins may be naturally derived or artificially synthesized.

Preferably, the lectin used in the present invention can be BC2LCN lectin or a variant thereof. BC2LCN lectin is the N-terminal domain of BC2L-C protein from Gram-negative bacteria (Burkholderia cenocepacia). A recombinant protein (rBC2LCN lectin) obtained by expressing this BC2LCN lectin in E. coli can be suitably used. rBC2LCN lectin is an undifferentiated 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 by transformed bacteria, and a specific method for its preparation is described, for example, in International Publication WO2016/147514. Furthermore, the BC2LCN lectin or its variant includes a BC2LCN lectin modified with any tag peptide known in the art.

In the second surface modifier, a hydrophilic chain and/or a linker may be present between the cell-binding protein and the azide group. As for the types of these hydrophilic chains and linkers, those mentioned above for the first surface modifier can be used. For these connections, as described above, for example, covalent bonds such as amide bonds, ester bonds, ether bonds, thioether bonds, carbamate bonds, thiocarbamate bonds, triazole bonds, and urea bonds can be used. In addition, each site may be connected in the same different binding manners or in different binding manners.

In a preferred embodiment, the azide groups in the second surface modifier are present at a ratio of 1 to 5, preferably 1 to 2.5, per molecule of cell-binding protein. It is suitable to use this abundance ratio of azide groups when immobilizing target cells by photopatterning surface modification as described below.

Addition of the second surface modifier onto the substrate can typically be performed by bringing the substrate into contact with a solution containing the second surface modifier. Conditions such as the solvent and contact time in the solution are the same as those for the first surface modifier.

1-3. Process (C)
As described above, in step (C), the connecting group N in the first surface modifying agent is reacted with the azide group in the second surface modifying agent, so that the second surface modifying agent is added to the upper layer of the first surface modifying agent. This is a step of covalently linking the surface modifiers. Thereby, a cell immobilization substrate having the cell-binding protein of the second surface modifier on the outermost surface can be obtained. Note that the present invention also relates to a cell immobilization substrate having a surface modified with the first and second surface modifiers obtained by the production method.

1-4. Photopatterning Step In a preferred embodiment of the production method of the present invention, a cell immobilization substrate modified with a cell-binding protein at a desired location can also be provided using photopatterning technology. This can be achieved by using a functional group that becomes reactive with the azide group upon light irradiation as the linking group N in the first surface modifier, and by linking the second surface modifier only to the light irradiated region. It can be carried out.

More specifically, the manufacturing method of the present invention can further include, before step (B), a step of irradiating the surface of the base material modified with the first surface modifier with light of a specific wavelength. Thereby, it is possible to obtain a cell immobilization substrate having a patterned surface modification in which the cell-binding protein is present only in a specific surface region.

For example, as shown below, when using a DBCO precursor with a protecting group as the linking group N in the first surface modifier, DBCO is generated when the first surface modifier is irradiated with 360 nm light. Then, it becomes a structure that can be bonded to the azide group of the second surface modifier by Huisgen cycloaddition reaction under physiological conditions.

This allows cell-binding proteins to be modified only in the area irradiated with light, so by setting the irradiation area according to the desired pattern, cell immobilization ability can be expressed only in the specific patterned area. becomes possible.

The wavelength of the light to be irradiated may be determined depending on the type of photoresponsive cell immobilizing agent, and is usually irradiated with light having a wavelength in the range of 157 to 600 nm, preferably around 250 to 450 nm. The light can be ultraviolet light. As a light source, sunlight, electric lamp light such as a mercury lamp, laser light (semiconductor laser, solid-state laser, gas laser), light emission from a light emitting diode, light emission from an electroluminescent element, etc. can be used. The method of light irradiation can be to uniformly irradiate the surface of the substrate with light from a light source through an appropriate filter if necessary, or to expose a pattern of a desired shape using a so-called photomask. good. Alternatively, light may be focused using a lens or mirror and irradiated onto a fine shape. Alternatively, scanning exposure may be performed using a focused light beam. In the case of pattern exposure, contact exposure may be performed, which is an exposure format in which a photomask and a base material are brought into contact with each other for exposure. Alternatively, proximity exposure may be performed, which is a non-contact exposure method in which the gap between the photomask and the base material is set to about several μm to several tens of μm. Furthermore, a projection exposure method (maskless exposure method) in which an image produced using a liquid crystal or a digital mirror device is projected onto the work surface may be used. The energy for light irradiation is sufficient as long as the surface of the substrate modified with the photoresponsive cell immobilizing agent can exhibit the function of immobilizing cells, and is usually 0.001 to 1000 J/cm ² and 0. .01 to 100 J/cm ² is preferable.
It is.

The shape of the pattern is not particularly limited, but may be, for example, in a form that allows cells to 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 where cells are immobilized, and even one point is included in "patterning."

2. Cell immobilization/recovery method of the present invention In another aspect, the present invention provides a method for selectively immobilizing target cells using the cell immobilization substrate surface-modified with the first and second surface modifiers. It also relates to the method of collection.

More specifically, the cell immobilization method of the present invention includes contacting the cell immobilization substrate with a solution containing predetermined target cells to immobilize the target cells on the surface of the cell immobilization substrate. including the step of Further, in the cell recovery method of the present invention, the immobilized target cells are separated and recovered from the cell immobilization substrate by treating the surface of the cell immobilization substrate with a lactose solution or an enzyme solution. including the step of

As a place for performing these methods, a cell immobilization substrate can be placed in a microchannel. The types of target cells are as described above, preferably stem cells, more preferably iPS cells.

The sugar solution and enzyme solution used in the cell recovery method competitively weaken the bond between the target cells immobilized on the substrate and cell-binding proteins, and release the target cells from the substrate surface under mild conditions. It is for. Examples of sugar solutions include fucose solutions, and examples of enzyme solutions include cell detachment solutions such as Accutase (registered trademark), trypsin/EDTA solutions, and protease solutions.

As described above, by optically patterning the surface of the substrate, target cells can be immobilized and recovered in a specific region. Furthermore, by providing a plurality of spot-shaped modification regions for immobilizing one cell, it becomes possible to immobilize and collect target cells at the single cell level. Note that the term "single cell level" is not limited to literally meaning one cell, but can also include cases such as 2 to 10 cells.

3. Kit of the Invention In a further aspect, the present invention also relates to a surface modification agent and a kit for use in surface modification of a substrate for cell immobilization.

The surface modifying agent used for surface modification of the substrate for cell immobilization corresponds to the above-mentioned first surface modifying agent, and as already mentioned, the surface modifying agent is a substrate that can be bonded to the surface of the substrate. It includes a bonding portion L, a hydrophilic chain M, and a linking group N that can be covalently bonded to an azide group.

Further, the kit of the present invention stores compounds corresponding to the first surface modifier and the second surface modifier.

Preferred embodiments and specific examples of the first surface modifier and the second surface modifier are as explained in the manufacturing method of the present invention above.

The reagents included in the kit include reagents normally used in this field, such as buffers, reaction promoters, stabilizers such as sugars, proteins, salts, and surfactants, and preservatives, which may coexist. It may contain substances that do not inhibit the stability of the reagent or the like and do not inhibit the reaction between the surface modifier and the target cells. Further, the concentration can be appropriately selected from the concentration range commonly used in this field.

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

Example 1: Synthesis of DBCO-containing surface modifier (first surface modifier)
The following compounds 1 and 2 having DBCO as a linking group capable of covalently bonding to an azide group were synthesized. These compounds correspond to the first surface modifier.

<Synthesis of compound 1>
Compound 1 was synthesized according to the synthesis scheme below. A hydrophilic polymer (polyethylene glycol: PEG) that can suppress non-specific adhesion of cells has a reactive group on both ends for modifying the substrate surface and a DBCO group that binds to an azide group by Heusgen cycloaddition reaction. and were introduced respectively.

[Compound 4]
Dry up a 100 mL three-neck eggplant flask by heating with a heat gun, add magnesium (2.4 g, 98.7 mmol, 13.1 eq), replace with Ar, and add 5 mL of dry THF and 1,2-dibromoethane (100 μL, 1.17 mmol, 0.16 eq) was added. Add 3-methoxybenzyl chloride (3.0 mL, 21 mmol, 2.7 eq) and 30 mL of dry THF to a dropping funnel connected to a three-necked eggplant flask, dry up together, and replaced with Ar, and stir in an ice bath for 3.5 hours. dripped. After drying and replacing with Ar, the reaction solution was transferred to a 100 mL two-neck eggplant flask containing Compound 3 (0.92 mL, 7.52 mmol, 1 eq), and refluxed at 71°C for 2 hours. Disappearance of compound 3 was confirmed by TLC (chloroform, Rf: compound 3 = 0.66, imine intermediate = 0.51). The reaction solution was transferred to a 100 mL two-necked eggplant flask containing NaBH ₄ (1.6016 g, 42.34 mmol, 5.63 eq), dried up, replaced with Ar, and added with 30 mL of dry MeOH, and stirred.

After 48 hours, the completion of the reaction was confirmed by TLC (Hexane/Acetone = 2/1, Rf: imine = 0.63, amine = 0), and after distilling off the solvent, 30 mL of pure water was added and the mixture was diluted with 50 mL of CH ₂ Cl ₂ Extracted 3 times. At this time, the aqueous phase became an emulsion, so it was filtered through Celite. The extracted organic phase was dried with NaSO ₄ , filtered with suction and the solvent was distilled off. Succinic anhydride (0.9641 g, 9.634 mmol, 1.28 eq) was placed in a beaker, dissolved in 20 mL of acetonitrile, and added to the extracted organic layer. Furthermore, DMAP (0.1915 g, 1.567 mmol, 0.21 eq) was added. Approximately 2 days later, I tried to confirm the disappearance of the raw material (amine) by TLC, but the DMAP spot was near the origin and could not be identified, so I back-extracted it 3 times with 50 mL of pure water to remove some of it, and the origin was removed. The spots (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) to obtain Compound 4 as a pale yellow transparent oil. Identification was performed using ¹ H-NMR (DMSO-d ₆ ) and ESI-MS (negative). The yield was 1.724 g, yield 64.1%.

[Compound 5]
Put AlCl ₃ (1.523 g, 11.43 mmol, 4.04 eq) into a 200 mL two-necked eggplant flask, dry up, replace with Ar, add dry DCM 30 mL, and tetrachlorocyclopropene (0.36 mL, 2.94 mmol, 1.04 eq). The mixtures were added sequentially and stirred at room temperature for 10 minutes, then cooled to -20°C. Compound 4 (0.9989 g, 2.827 mmol, 1 eq) was placed in another 100 mL eggplant flask, dried up, replaced with Ar, and 20 mL of dry DCM was added and cooled to -20°C with stirring.
A solution containing compound 4 was transferred to a solution containing tetrachlorocyclopropene by cannulation over 10 minutes, stirred at -20°C for 5 hours and 30 minutes, and further stirred at room temperature for 2 hours. After confirming the disappearance of the raw material by TLC (methanol/dichloromethane = 1/5, Rf: Compound 4 = 0.45), 10 mL of pure water was added and the mixture was stirred vigorously.

When acetone was added to the oil obtained by concentrating the reaction solution, an off-white precipitate was formed. The supernatant was removed by decantation, and acetone was added again for washing three times in total, and the precipitate was collected by suction filtration to obtain Compound 5 as an off-white solid. Analysis by ¹ H-NMR (DMSO-d ₆ , 600 MHz) showed that the yield was 229 mg, 19.8%.

[Compound 6]
Put 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) into a 10 mL two-necked eggplant flask and dry. The mixture was replaced with Ar, and 5 mL of dry DCM was added to suspend the mixture and stirred to start the reaction. After about 18 hours, the disappearance of the raw material was confirmed by TLC (methanol/dichloromethane = 1/10, Rf: Compound 5 = 0.11, Compound 6 = 0.42, detected by UV and bromocresol green), and the solvent was distilled off to 2 mL. was suspended in acetone and washed with ultrasonic waves. The precipitate was collected by suction filtration and washed on a filter paper with 8 mL of acetone to obtain Compound 6 as a light brown solid. The yield was 86.0 mg, 68%.

[Compound 7]
Compound 6 (39.51 mg, 7.832×10-5 mol, 2.52 eq), HOOC-PEG _n -NH ₂ (MW: 3400, manufactured by NOF Corporation, product name “Sunbright PA-034HC”) was placed in a 10 mL two-neck eggplant flask. ) (105.81 mg, 3.112×10-5 mol, 1 eq), dry up, replace with Ar, add 3 mL of dry DMF and dry TEA (0.04 mL, 2.9×10-4 mol, 9.2 eq). The mixture was stirred and the reaction started. After 40 hours, the disappearance of the raw materials was confirmed by TLC (methanol/dichloromethane = 1/5, Rf: PEG = 0.29, Compound 7 = 0.32, detected with ninhydrin), and the solvent was distilled off to precipitate ether (diethyl ether 40 mL). , crude in dichloromethane (1 mL, -15°C, 15000 G, 10 min), air-dried, and then dissolved in approximately 10 mL of pure water and dialyzed. Dialysis was performed for 31 hours and lyophilized to obtain Compound 7 as a light brown solid. Identification was performed by ¹ H-NMR (DMSO-d ₆ ) and MALDI-TOF-MS (Dithranol & NaCl, positive), and the yield was 100.8 mg, 85%.

[Compound 8]
Compound 7 (19.58 mg, 5.17°10-6 mol) was placed in a cent tube, dissolved in 1 mL of DMSO- _d6 , and irradiated with 8 J of light using a light irradiator. When the reaction was confirmed by ¹ H-NMR (DMSO-d ₆ ), some raw material was found, so I used a hand-held UV light to shine light on it until the raw material disappeared. Identification was performed by ¹ H-NMR (DMSO-d ₆ ), and pure water was added and freeze-dried to obtain Compound 8 as a light brown solid. The yield was 19.0 mg, 97%.

[Compound 1]
NHS (4.06 mg, 3.53×10-5 mol, 6.4 eq) and DCC (12.75 mg, 6.65×10-5 mol, 12 eq) were placed in a 10 mL two-necked eggplant flask, dried up, and replaced with Ar. Compound 8 (19.0 mg, 5.01 x 10-6 mol, 1 eq) dissolved in 3 mL of dry DCM was added and stirred to start the reaction. After about 26 hours, the completion of the reaction was confirmed by TLC (methanol/dichloromethane = 1/5, Rf: Compound 8 = 0.58), the solvent was distilled off, the solution was redissolved in about 1 mL of DCM, and the mixture was dissolved in 40 mL of diethyl ether. Ether precipitation (-15°C, 15000 G, 10 min) was performed. The supernatant was removed and air-dried, 3 mL of DCM was added and filtered through a cotton plug, and the solvent was distilled off to obtain Compound 1 as a light brown solid. The yield excluding the remaining DCC urea was 12.8 mg, which was 66%. Identification was performed by ¹ H-NMR (DMSO-d ₆ ).

<Synthesis of compound 2>
Similarly, Compound 2 was synthesized using the following synthesis scheme. The synthesis of compounds 3 to 7 is the same as the synthesis of compound 1 above.

[Compound 2]
Compound 7 (20.99 mg, 5.54×10-6 mol, 1 eq), NHS (4.06 mg, 3.53×10-5 mol, 6.4 eq), DCC (12.75 mg, 6.65×10- 5 mol, 12 eq) was added, dried up, and replaced with Ar. 3 mL of dry DCM was added and stirred to start the reaction. After about 26 hours, the completion of the reaction was confirmed by TLC (MeOH/DCM = 1/5, Rf: Compound 7 = 0.45, Compound 2 = 0.58), the solvent was distilled off (I forgot to filtrate with a cotton plug), and the reaction was repeated. It was redissolved in about 1 mL of DCM and ether precipitated with 40 mL of diethyl ether (-15°C, 15000 G, 10 min). The supernatant was removed, air-dried, 3 mL of DCM was added, the mixture was filtered through a cotton plug, and 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%. Identification was performed using ¹ H-NMR (DMSO-d ₆ ) and MALDI-TOF MS (positive, Matrix: Dithranol and NaCl).

Example 2: Synthesis of lectin-containing surface modifier (second surface modifier)
Next, a compound in which azidation was introduced into lectin (rBC2LCN) was synthesized using a molecule (NHS-PEG4-azide) in which a lectin-binding group (NHS), PEG (polymerization 4), and an azide group were bonded in this order.

The solvent of the rBC2LCN aqueous solution was replaced with a borate buffer (pH 8.3) by dialysis (MWCO: 3500), and the concentration was calculated from the absorbance of light at 280 nm, and it was found to be 42.6 μM. To this rBC2LCN aqueous solution, NHS-PEG4-azide solution (100 mM) was added in an amount of 5 equivalents, stirred, and then allowed to stand at 4°C for about 23 hours. Thereafter, purification and solvent replacement were performed by dialysis (MWCO: 3500) to obtain an azide lectin (rBC2LCN-azide) aqueous solution. In order to confirm the success or failure of modification of the azide group, a gel shift assay was performed. A DMSO solution of a molecule in which DBCO was bonded to the end of PEG (average polymerization number 73) was mixed in an amount of 100 equivalents to rBC2LCN-azide, and reacted for about 24 hours. Thereafter, band movement was confirmed by polyacrylamide gel electrophoresis. As a result, in addition to the unmodified band, multiple bands were observed on the high molecular weight side (Figure 1). Since each corresponds to a modification amount of 1 to 4, the average modification rate of the azide group to the lectin was calculated to be 1.1.

Example 3: Lectin modification on the substrate surface and capture of iPS cells The DBCO-containing surface modifier (compound 2) synthesized in Example 1 was dissolved in ethanol (20 μL), and collagen was aminated by physical adsorption. It was modified by applying 90 μL to the entire surface of the glass substrate and air drying. A commercially available microchannel (manufactured by ibidi GmbH, product name "Sticky-dlide VI") was attached, and the surface was washed with ultrapure water. Thereafter, the area to be irradiated with light was limited to a line shape using a photomask, and 0.1 J/cm ² of 365 nm light was irradiated. A PBS aqueous solution of the lectin-containing surface modifier (rBC2LCN-azide: average modification rate of azide group is 1.1) synthesized in Example 2 was reacted at 25 μM for 105 minutes. After washing the surface with PBS to remove unreacted rBC2LCN-azide, a suspension of iPS cells (1×10 ⁷ cells/mL) stained with Calcein-AM was applied for 10 minutes. After washing away uncaptured cells with PBS, the substrate surface was observed using a fluorescence microscope. A schematic diagram of the experimental procedure is shown in FIG. 2.

As a result, it was confirmed that cells were captured in the area irradiated with light. On the other hand, no cell capture was observed in the area where light was not irradiated, indicating that cells were captured via rBC2LCN-azide only in the light irradiation area (FIGS. 3a and 3b).

On the other hand, when similar experiments were conducted using rBC2LCN-azide (highly azide lectin) with an average amount of 3.0 azide group modifications, no cell capture was observed even in the light irradiation region, indicating that the amount of azide group modification is important. It was also shown that there is (Figure 4).

Example 4: Selective capture and recovery of iPS cells in the presence of contaminant cells The results of Example 3 showed that it was possible to selectively modify the lectin (rBC2LCN-azide) in the light irradiation area and capture iPS cells. Therefore, we next verified that iPS cells could be selectively captured on the substrate from a mixed suspension of multiple types of cells.

Dissolve the DBCO-containing surface modifier (compound 2) synthesized in Example 1 in ethanol (20 μL), apply 90 μL to the entire surface of the glass substrate surface that has been aminated by physically adsorbing collagen, and modify by air drying. did. A commercially available microchannel (manufactured by ibidi GmbH, product name "Sticky-dlide VI") was attached, and the surface was washed with ultrapure water. Thereafter, the area to be irradiated with light was limited to a line shape using a photomask, and 0.1 J/cm ² of 365 nm light was irradiated. A PBS aqueous solution of the lectin-containing surface modifier (rBC2LCN-azide) synthesized in Example 2 was reacted at 25 μM for about 2 hours. After washing the surface with PBS to remove unreacted rBC2LCN-azide, iPS cell suspension (Calcein-AM staining, 1 × 10 ⁷ cells/mL) and Jurkat cells (CytoRed staining, 1 × 10 ⁷ cells/mL) were added. mL) at a ratio of 1:1 and allowed to act for 10 minutes. Here, since Jurkat cells do not have sugar chains to which rBC2LCN binds on their cell surface, they are not captured on the rBC2LCN-azide modified surface in principle. After washing away uncaptured cells with PBS, the substrate surface was observed using a fluorescence microscope.

The results are shown in Figures 5a-b. Figure 5a is a fluorescence microscopy image of cells immobilized when a mixed suspension containing two types of cells was applied to a substrate modified with a lectin-containing surface modifier (rBC2LCN-azide); Figure 5b This is a fluorescence microscopy image of cells immobilized when a mixed suspension containing two types of cells was applied to a substrate modified with azidated PEG lipid (Oleyl-PEG-azide) that does not have lectin. It is. The molecular structure of Oleyl-PEG-azide is shown in Figure 5c.

As a result, only iPS cells were selectively captured on the substrate in the light irradiated area (Fig. 5a). In addition, as a target experiment, we conducted a similar experiment using azidated PEG lipid (Oleyl-PEG-azide: Figure 5c) that does not have a lectin instead of rBC2LCN-azide, and found that iPS cells and Jurkat cells Both were captured on the substrate (Fig. 5b).

Subsequently, the same substrate was treated with Accutase (registered trademark), a cell detachment enzyme, and 0.25% trypsin/EDTA in this order at 37°C for a total of 10 minutes. After washing the substrate with PBS, the surface of the substrate was observed using a fluorescence microscope. The results are shown in Figures 5d-g. Figure 5d is a fluorescence microscopy image of cells captured on the substrate surface modified with rBC2LCN-azide after treatment with protease; Figure 5e is a fluorescence microscopy image of cells captured on the substrate surface modified with rBC2LCN-azide; Figure 3 is a fluorescence microscopy image of cells on a substrate after treatment of cells captured on the surface with proteolytic enzymes. FIGS. 5f and 5g are graphs obtained by quantifying changes in cell density before and after enzyme treatment on substrates modified with rBC2LCN-azide and Oleyl-PEG-azide using image analysis software.

As a result, it was observed that many of the cells captured using rBC2LCN-azide had detached from the substrate surface (FIGS. 5d and 5f). On the other hand, almost all of the cells captured using Oleyl-PEG-azide remained on the substrate (FIGS. 5e and 5g).

These results demonstrate that iPS cells can be selectively captured on a substrate from among multiple cell suspensions by using a lectin-containing surface modifier (rBC2LCN-azide), and that This demonstrates that it is possible to collect iPS cells from the substrate by processing under the following conditions.

Example 5: Selective capture of iPS cells in the presence of cells differentiated from iPS cells Next, iPS cells were selectively captured on a substrate from a mixed suspension with neural progenitor cells differentiated from iPS cells. We verified that it can be captured.

STEMdiff SMADi Neural Induction Kit (ST-08581, STEMCELL technologies) was used to induce differentiation of iPS cells into neural progenitor cells. A culture dish coated with Poly-L-Ornithine was modified with 10 μg/mL laminin, and iPS cells (1×10 ⁵ /cm ² ) were seeded therein. STEMdiff Neural Induction Medium (STEMdiff Neural Induction Supplement, 10 μM Y-27632 added) was used as the medium, and the culture was performed for 3 days with medium exchange every day.

After modifying the substrate surface with a lectin-containing surface modifier (rBC2LCN-azide) in the same manner as in Example 4, iPS cells (1×10 ⁶ cells/mL) and neural progenitor cells (CytoRed) differentiated from iPS cells were modified. A cell suspension mixed at a ratio of 1:1 with 1×10 ⁶ cells/mL) was allowed to act for 10 minutes. Here, the differentiated neural progenitor cells have lost sugar chains to which rBC2LCN binds from the cell surface layer, and therefore are not captured by the rBC2LCN-modified surface in principle. Uncaptured cells were washed away with PBS and reacted with rBC2LCN-FITC (fluorescein isothiocyanate), a reagent that fluorescently labeled rBC2LCN, for 15 minutes, then washed with PBS, and the substrate surface was observed with a fluorescence microscope (Figures 6a and 6a). b). Figure 6a is a fluorescence microscopy image of cells captured when a mixed suspension of iPS cells and iPS cell-derived neural progenitor cells was applied to a substrate modified with a lectin-containing surface modifier (rBC2LCN-azide). Yes; Figure 6b shows the results when a mixed suspension of iPS cells and iPS cell-derived neural progenitor cells was applied to a substrate modified with azidated PEG lipid (Oleyl-PEG-azide) that does not have lectin. Fluorescence microscopy images of captured cells. FIG. 6c is a graph quantified by image analysis of the cell density in the light irradiated area on the substrate modified with rBC2LCN-azide and Oleyl-PEG-azide.

As a result, on the surface modified with a lectin-containing surface modifier (rBC2LCN-azide), only undifferentiated iPS cells stained with rBC2LCN-FITC were selectively captured on the substrate in the light irradiated area (FIG. 6c). On the other hand, both iPS cells and neural progenitor cells were captured on the Oleyl-PEG modified surface used as a control experiment as in Example 4 (FIG. 6c).

Claims (21)

A method for producing a cell immobilization substrate for immobilizing predetermined target cells on the substrate, the method comprising:
(A) a step of modifying the whole or part of the surface of the base material with a first surface modifier, the first surface modifier including a compound having the structure shown below,
L-M-N
In the formula, L is a substrate binding part that can be bonded to the surface of the substrate, M is a hydrophilic chain, and N is a linking group that can be covalently bonded to an azide group;
(B) adding onto the substrate a second surface modifier that includes a cell-binding protein that can selectively bind to the target cell and has a structure in which an azide group is linked to the cell-binding protein; and (C) modifying the cell-binding protein on the surface of the base material by forming a covalent bond between the linking group N in the first surface modifying agent and the azide group in the second surface modifying agent. The manufacturing method includes the step of obtaining a base material for cell immobilization. The manufacturing method according to claim 1, further comprising, before the step (B), irradiating the surface of the base material modified with the first surface modifier with light of a specific wavelength. The manufacturing method according to claim 1, wherein the cell immobilization substrate has a patterned surface modification in which the cell-binding protein is present only in a specific surface region. The production method according to claim 1, wherein the second surface modifier has 1 to 2.5 azide groups per molecule of the cell-binding protein. The manufacturing method according to claim 1, wherein the connecting group N includes an alkynyl group or a group in which a protecting group is introduced into an alkynyl group. The manufacturing method according to claim 1, wherein the linking group N has a structure containing dibenzocyclooctyne (DBCO) or a precursor thereof. The manufacturing method according to claim 1, wherein the hydrophilic chain M includes a hydrophilic polymer. The manufacturing method according to claim 1, wherein the hydrophilic chain M contains polyalkylene glycol. The manufacturing method according to claim 1, wherein the base material bonding portion L has a substituent that can be bonded to the surface of the base material through a covalent bond. The manufacturing method according to claim 1, wherein the target cells are stem cells. The manufacturing method according to claim 10, wherein the stem cells are induced pluripotent stem cells (iPS cells) or embryonic stem cells (ES cells). The manufacturing method according to claim 1, wherein the cell-binding protein is a lectin or an antibody. The manufacturing method according to claim 12, wherein the lectin is BC2LCN lectin or a variant thereof. A method for immobilizing cells on a cell immobilization substrate obtained by the production method according to any one of claims 1 to 13, comprising:
The method includes the step of contacting the cell immobilization substrate with a solution containing predetermined target cells, and immobilizing the target cells on the surface of the cell immobilization substrate. A method for collecting cells fixed by the method according to claim 14, comprising:
The method includes the step of separating and collecting the immobilized target cells from the cell immobilization substrate by treating the surface of the cell immobilization substrate with a sugar solution or an enzyme solution. A surface modifier for a substrate for immobilizing predetermined target cells, the agent comprising:
Structure below:
L-M-N
(In the formula, L is a substrate binding part that can be bonded to the surface of the substrate, M is a hydrophilic chain, and N is a linking group that can be covalently bonded to an azide group.)
The surface modifier comprises a compound represented by: The surface modifier according to claim 16, wherein the linking group N has a structure containing dibenzocyclooctyne (DBCO) or a precursor thereof. The surface modifier according to claim 16, selected from compounds having the following structure:

A kit for use in surface modification of a substrate for cell immobilization, comprising the surface modification agent according to any one of claims 16 to 18, and
A kit containing a compound having a structure in which an azide group is linked to a cell-binding protein that can selectively bind to target cells. The kit according to claim 19, wherein the cell-binding protein is a lectin or an antibody. A base material for cell immobilization, comprising:
Structure below:
L-M-N
(In the formula, L is a substrate binding part that can be bonded to the surface of the substrate, M is a hydrophilic chain, and N is a linking group that can be covalently bonded to an azide group.)
A first surface modifier comprising a compound represented by; and
The base material has a surface modified with a second surface modifier, which contains a cell-binding protein capable of selectively binding to a predetermined target cell, and has a structure in which an azide group is linked to the cell-binding protein.

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