WO2019088258A1 - Photodegradable cell immobilization agent - Google Patents

Abstract

[Problem] To develop a photodegradable cell immobilization agent having superior cell immobilization ability. [Solution] This photodegradable cell immobilization agent is for immobilizing a predetermined target cell onto a base material, the agent being characterized by comprising a hydrophobic chain capable of interacting with the target cell, a hydrophilic chain capable of being arrayed into a monomolecular film on a surface of the base material, and a hydrophilic linker and a photodegradable linker which link the hydrophobic chain and the hydrophilic chain together, wherein the hydrophilic linker is positioned between the hydrophobic chain and the photodegradable linker.

Description

Photodegradable cell fixing agent

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

In recent years, in a wide range of fields from basic research in cell biology to regenerative medicine, a technology for analyzing cells at the single cell level and sorting them at high throughput is required. For example, circulating cancer cells (Circulating Tumor Cells; CTCs) in the blood have a very low proportion, but sorting and analysis of CTCs enables elucidation of the mechanism of canceration and evaluation of therapeutic effects, etc. Is expected to be In addition, in the regenerative medicine field, when differentiating and using ES cells and iPS cells, it is necessary to appropriately remove undifferentiated cells which may be transformed into cancer. Furthermore, selective recovery of hybridomas with high antibody production ability contributes to efficient production of antibody pharmaceuticals. Thus, the cell separation / sorting technology at the single cell level is expected to be applied in a wide range, and the development of the technology is required.

Conventional cell separation / sorting techniques can be classified as in the following 1) to 5).
1) Method of centrifugation using differences in cell size and specific gravity 2) Method of fluorescently labeling cells and separation using a flow cytometer 3) Arraying cells using microwells and separation while observing Method 4) Method of fixing cells with a photolytic gel and dissolving and separating the gel by light irradiation while observing 5) Fixing cells by light irradiation while arranging cells using a photodegradable cell immobilization material Method of decomposing and separating

However, although the method (1) is a simple cell separation method, there are problems such as low separation accuracy and unsuitable for selection of rare cells. The method (2) is the most commonly used cell sorting method because of its high throughput, but it can not simultaneously observe differences between cells because it can not observe multiple cells simultaneously at the same time, and it can not follow changes over time. There is a problem called. The method of (3) has recently attracted attention in that it can overcome the problems of the method of (2). However, problems remain in the method of recovering arrayed cells. For example, in the method using a micromanipulator, the throughput is lowered because each cell is absorbed by a capillary (eg, Patent Document 1). Other techniques for extracting cells electrochemically and physicochemically have been developed, but have the disadvantage that the cells are directly stressed.

In addition, the method (4) has recently attracted attention in that it is highly likely to overcome the separation speed, which is the problem of the method (3). An aqueous solution containing cells is gelled using a photodegradable crosslinking agent, and the gel can be dissolved by light irradiation, and only desired cells can be recovered conveniently and rapidly (eg, Patent Document 2). However, in order to immobilize individual cells without contacting them in the lateral direction and stacking them in the vertical direction, the cell density must be lowered, and the number of cells that can be observed and separated at one time is extremely small. There is a drawback.

On the other hand, the method (5) is advantageous in that the low cell density, which is the problem of the method (4), can be improved. For example, the present inventors arrange and fix cells in a microchannel with already photolyzable polyethylene glycol (PEG) lipid, and when the cells to be collected are subjected to light irradiation to apply flux, the cells It has been found that it is possible to selectively detach from the substrate (Non-patent Document 1). However, even in such a method, the immobilizing power of the cells is low, and when one cell is separated, cells not irradiated with light are also detached by the shear stress of the microfluidic and can not be used for cell separation. There was a problem of that. Therefore, a strong cell immobilization power photolytic cell immobilization agent capable of sufficiently immobilizing single cells is essential for cell isolation.

JP, 2010-14438, A JP 2014-226089 A

S. Yamaguchi, et al, Angew. Chem. Int. Ed. 2012, 51, 128-131

The photocleavable cell fixing agent of Non-Patent Document 1 developed by the inventors until now is a compound in which polyethylene glycol (PEG) and a lipid are linked via a photocleavable linker, and this molecule is used as a substrate In this case, since the lipid moiety interacts with the lipid bilayer of cells, it is possible to immobilize any cells, whether adherent cells or non-adherent cells, on a substrate. However, if the spot size is reduced and cells are immobilized one by one, the flow rate required to release the cells allows 60 to 80% of the cells to be released in a few minutes without light irradiation. It will Then, this invention makes it a subject to develop the photodegradable cell fixing agent excellent in cell immobilization power, in order to compensate the fault of the conventional cell sorting method.

As a result of intensive studies to solve the above problems, the present inventors have found that a specific length between the photocleavable site in the structure of the conventional photodegradable PEG lipid and the hydrophobic site that binds to the target cell is By inserting a hydrophilic region having a diameter, it is possible to strengthen the cell immobilization power, stably immobilize a substance covered with any cell or lipid membrane, and selectively recover it by light irradiation. It can be found that the present invention has been completed.

（式中、ｍは、２より大きい自然数である。）
＜５＞ｎが、４～４０の自然数である、上記＜４＞に記載の光分解性細胞固定化剤；
＜６＞前記疎水性鎖が、置換基を有していてもよい飽和又は不飽和の炭化水素鎖である、上記＜１＞～＜５＞のいずれかに記載の光分解性細胞固定化剤；
＜７＞前記親水性鎖が、親水性ポリマーを含む、上記＜１＞～＜６＞のいずれかに記載の光分解性細胞固定化剤；
＜８＞前記親水性鎖が、ポリアルキレングリコールを含む、上記＜１＞～＜６＞のいずれかに記載の光分解性細胞固定化剤；
＜９＞前記光分解性リンカーが、２－ニトロベンジル骨格、クマリン－４－イルメチル骨格、フェニルカルボニルメチル骨格又は７－ニトロインドリノカルボニル骨格を有する二価の基である、上記＜１＞～＜８＞のいずれかに記載の光分解性細胞固定化剤；
＜１０＞前記親水性鎖及び親水性リンカーがいずれも、ポリエチレングリコールを含む、上記＜１＞～＜９＞のいずれかに記載の光分解性細胞固定化剤；
＜１１＞前記親水性鎖の末端に、前記基材の表面と共有結合により結合し得る置換基を有する、上記＜１＞～＜１０＞のいずれかに記載の光分解性細胞固定化剤；及び
＜１２＞以下の構造を有する、上記＜１＞に記載の光分解性細胞固定化剤

（式中、ｍは、２～４０の自然数であり、ｎは、４５～５００の自然数である。）
を提供するものである。 That is, in one aspect, the present invention
<1> A photocleavable cell fixing agent for fixing a predetermined target cell on a substrate, the hydrophobic chain capable of interacting with the target cell, in the form of a monolayer on the surface of the substrate It has a hydrophilic chain that can be arranged, and a hydrophilic linker that links the hydrophobic chain and the hydrophilic chain, and a photodegradable linker, and the hydrophilic linker is between the hydrophobic chain and the photodegradable linker. Said photodegradable cell fixing agent characterized in that it is arranged in
<2> The photodegradable cell fixing agent according to <1>, wherein the hydrophilic linker comprises a hydrophilic polymer;
<3> The photodegradable cell fixing agent according to <1>, wherein the hydrophilic linker comprises a polyalkylene glycol;
<4> The photodegradable cell fixing agent according to <1>, wherein the hydrophilic linker has the following partial structure;

(Wherein, m is a natural number greater than 2)
The photolyzable cell fixing agent according to the above <4>, wherein <5> n is a natural number of 4 to 40;
<6> The photodegradable cell fixing agent according to any one of <1> to <5>, wherein the hydrophobic chain is a saturated or unsaturated hydrocarbon chain which may have a substituent. ;
<7> The photodegradable cell fixing agent according to any one of the above <1> to <6>, wherein the hydrophilic chain comprises a hydrophilic polymer;
<8> The photodegradable cell fixing agent according to any one of the above <1> to <6>, wherein the hydrophilic chain comprises a polyalkylene glycol;
<9> The above <1> to <10, wherein the photocleavable linker is a divalent group having a 2-nitrobenzyl skeleton, a coumarin-4-ylmethyl skeleton, a phenylcarbonylmethyl skeleton or a 7-nitroindolinoxycarbonyl skeleton. The photolyzable cell fixing agent according to any one of 8>;
<10> The photodegradable cell fixing agent according to any one of the above <1> to <9>, wherein the hydrophilic chain and the hydrophilic linker each contain polyethylene glycol;
<11> The photodegradable cell fixing agent according to any one of the above <1> to <10>, having a substituent which can be covalently bonded to the surface of the substrate at the end of the hydrophilic chain; And the photodegradable cell fixing agent according to <1>, having a structure of <12> or less

(Wherein, m is a natural number of 2 to 40, and n is a natural number of 45 to 500)
To provide

In another aspect, the present invention provides:
<13> A substrate for cell immobilization, having a surface modified with the photolytic cell immobilization agent according to any one of <1> to <12>above;
<14> The substrate for cell immobilization according to <13>, which has a patterned surface modification having a photolytic cell immobilization agent only in a specific surface area of the substrate; and <15> A step of modifying the entire substrate surface with the photolytic cell fixing agent according to any one of the above <1> to <13>, and the surface modification with the photolytic cell fixing agent is maintained only in a specific region A step of patterning the surface, wherein light is irradiated to a region other than the specific region on the surface of the substrate to cut the photodegradable linker moiety in the photodegradable cell fixing agent, The present invention provides a method for producing a cell immobilizing substrate comprising the

In yet another aspect, the invention provides:
The process which makes the solution containing a predetermined | prescribed target cell contact the base material for cell immobilization as described in <16> said <13> or <14>, and immobilizes the said target cell on the said base material for cell immobilization, the said The substrate for cell immobilization is irradiated with light, and the photocleavable linker moiety in the photolyzable cell immobilization agent on the surface of the substrate for cell immobilization is cleaved, whereby the immobilized target cells are isolated. The present invention provides a method for recovering cells, which comprises the steps of: separating and recovering from a substrate for cell immobilization.

According to the photodegradable cell immobilization agent of the present invention, the cell immobilization power can be enhanced, so that target cells can be stably immobilized and selectively recovered by light irradiation. In addition, the substrate for cell immobilization which has been surface-modified with the photolytic cell-fixing agent of the present invention can immobilize cells at a single cell level by patterning the surface modification region in advance by light irradiation, and a strong flow rate The cell pattern can be firmly maintained under

FIG. 1 is a schematic view showing the entire structure of the photodegradable cell fixing agent of the present invention. FIG. 2 is a schematic view of a substrate surface-modified with the photolytic cell fixing agent of the present invention and a conceptual view of the immobilization / recovery of cells. FIG. 3 is a schematic view showing a patterning step of surface modification in the substrate for cell immobilization of the present invention. FIG. 4 is a graph showing the cell survival rate for the photolytic cell immobilization agent of the present invention and a comparative example. FIG. 5 is a graph showing the light irradiation dependency of the cell survival rate by the photolytic cell fixing agent of the present invention. FIG. 6 is an image of a 1-cell microarray formed on the substrate for cell immobilization of the present invention. FIG. 6 (a) is a fluorescence image of an EGFP-expressing BaF3 cell array formed on a cell immobilization substrate coated with collagen as a covering layer. FIG. 6 (a) is a bright field image of an EGFP-expressing BaF3 cell array formed on a cell immobilization substrate coated with BSA as a coating layer. FIG. 7 is a fluorescence image before and after selective detachment of the cell array. FIG. 8 is a graph showing the cell detachment rate in light-irradiated spots and non-irradiated spots. FIG. 9 is an image of dendritic cells cultured on a substrate for cell immobilization.

Hereinafter, embodiments of the present invention will be described. The scope of the present invention is not restricted by these explanations, and it can be suitably changed and implemented in the range which does not spoil the meaning of the present invention also except the following examples.

1. Photodegradable Cell Fixing Agent The photodegradable cell fixing agent of the present invention is
(A) a hydrophobic chain having a function of interacting with a target cell to bind to the cell,
(B) hydrophilic chains which can be arranged in the form of a monomolecular film on the surface of a substrate,
Furthermore, it has (c) a hydrophilic linker and (d) a photocleavable linker as two linker sites for linking these hydrophobic chains and hydrophilic chains. And, it is characterized in that the hydrophilic linker (c) is disposed between the hydrophobic chain (a) and the photodegradable linker (d).

Therefore, as shown in FIG. 1, the entire structure of the photolytic cell fixing agent of the present invention is a structure in which these (a) to (d) are linked in the following order. The linkage of each site may 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 the photocleavable linker moiety (d) and the hydrophilic chain (b) is an amide bond or an ester bond from the viewpoint of permitting cleavage of the photodegradable linker moiety (d) by light irradiation. Is preferred.

In FIG. 2, the schematic diagram of the base material surface-modified by the photolytic cell fixing agent of this invention, and the conceptual diagram of immobilization and collection | recovery of a cell are shown. The photolytic cell fixing agent modifies the surface of the substrate by binding to the surface of the substrate directly at the end of the hydrophilic chain (b) or via a covering layer described later. The photocleavable cell fixing agent is preferably arranged in the form of a monolayer on the surface of the substrate. On the other hand, the hydrophobic chain (a) can bind to and capture a target cell by an interaction such as a hydrophobic interaction. Thereby, target cells can be immobilized on a specific region of the substrate surface. Thereafter, light irradiation is performed on a desired region on the substrate surface, whereby the target cells can be separated from the substrate surface and recovered by being decomposed with the photodegradable linker portion (d).

Here, "cells" can include animal cells, plant cells, insect cells, prokaryotic cells, fungal cells and the like, and generally do not adhere to or extend on the surface of a carrier such as a culture device, but in a suspended or precipitated state What is referred to as proliferating "suspended cells" (eg, blood cells) or "adhered cells" that adheres to or extends on the surface of the carrier are dispersed from the carrier with a suitable dispersing agent such as EDTA-trypsin, dispase, etc. and temporarily suspended. (Eg, fibroblasts detached from carrier by EDTA solution), and cells in a state of being adhered to carrier. Also included are organisms having a phospholipid bilayer membrane on the surface of liposomes, exosomes, bacteria, viruses, organelles, plant cells from which cell walls have been removed (protoplasts) and the like. Moreover, according to the photodegradable cell immobilization agent of the present invention, in addition to these, substances having lipids such as lipid-coated particles can also be immobilized.

The hydrophobic chain (a) is a site for interacting with a target cell and capturing the target cell. As such interaction, noncovalent interaction such as hydrophobic interaction can be used. Specifically, the hydrophobic chain (a) can bind to a target cell by hydrophobic interaction with a lipid moiety in a cell membrane or the like which is a lipid bilayer membrane.

The hydrophobic chain (a) is not particularly limited as long as it can bind to a target cell by hydrophobic interaction, but it may be a saturated or unsaturated hydrocarbon chain which may have a substituent. Examples of such hydrocarbon chains include, for example, a C _7-30 alkyl group (preferably a C _7-22 alkyl group), a C _6-14 aryl group, and a C _6-14 aryl C _7-30 alkyl group (preferably C 6 C). _6-14 aryl C _7-22 alkyl group), and C _7-30 alkyl C _6-14 aryl group (preferably C _6-14 aryl C _7-22 alkyl group) and the like. Preferably, adjacent carbon atoms may be linked by a C _7-30 alkyl group which may be linked by 1 to 3 unsaturated bonds, and adjacent carbon atoms may be linked by 1 to 3 unsaturated bonds C _7-22 alkyl group, or C _11-22 alkyl group in which adjacent carbon atoms may be linked by 1 to 3 unsaturated bonds, or adjacent carbon atoms are linked by 1 to 3 unsaturated bonds It may be a C _16-18 alkyl group which may be linked. More preferably, the hydrophobic chain (a) is a hexadecyl group, a heptadecyl group, an octadecyl (stearyl) group, a cis-9-hexadecenyl (palmitolayl) group, a cis-8-heptadecenyl group, a trans-8-heptadecenyl group, a trans -9-octadecenyl (elaidyl) group, cis -9-octadecenyl (oleyl) group, cis, cis-9,12-octadecadienyl (linolenyl) group, (9E, 12E, 15E) -octadeca-9, 12, It can be a 15-trienyl (erilide linolenyl) group. In particular, the oleyl group which is a part of the phospholipid which comprises a cell membrane is preferable. Furthermore, these hydrophobic chains may be substituted with any substituent, and may contain heteroatoms such as N, S, O and the like.

The hydrophilic chain (b) is preferably constituted by a hydrophilic polymer. As such a hydrophilic polymer, 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 an oxyalkylene unit having 2 to 4 carbon atoms, and one having an average polymerization number in the range of 2 to 500 (preferably 45 to 500) can be used. The hydrophilic polymer is preferably a biocompatible polymer, more preferably polyethylene glycol (PEG). The hydrophilic chain (b) may further have an optional substituent.

As described above, the hydrophilic chain (b) preferably has a functional group at its end for linking to the substrate surface by covalent bond or the like. As such a terminal functional group, for example, those shown below can be used (wherein, the arrow indicates the point of attachment to the hydrophilic chain (b)).

Preferably, the terminal functional group may be an active ester group such as N-hydroxysuccinimide (NHS), a carboxyl group, a silanol group, a disulfide group or a thiol group. As described later, when a coating layer such as collagen is used on the substrate surface, a functional group capable of binding to the coating layer can be used. For example, in the case of a collagen coating layer, an amino group in collagen Preferred is an active ester group which can be covalently bonded to, particularly preferably having an NHS group.

The hydrophilic linker (c) is disposed between the hydrophobic chain (a) and the photocleavable linker (d) as described above. It is a feature of the present invention that by inserting a hydrophilic linker (c) having a predetermined length, target cells can be more firmly immobilized as compared with conventional methods. The hydrophilic linker (c) is preferably constituted by a hydrophilic polymer. As the hydrophilic chain (b), as the hydrophilic polymer, polysaccharides such as the above polyalkylene glycol, polyvinyl alcohol, polyacrylic acid, polypeptide, polyacrylamide, dextran and the like, or glycolic acid derivatives and lactic acid derivatives, Polymers, copolymers and the like of p-dioxane derivatives can be used. The polyalkylene glycol is preferably a polymer of an oxyalkylene unit having 2 to 4 carbon atoms, and one having an average polymerization number in the range of 2 to 100 (preferably 4 to 40) can be used. The hydrophilic polymer is preferably a biocompatible polymer, more preferably polyethylene glycol (PEG).

More specifically, the hydrophilic linker (c) preferably has the following partial structure having a repeating unit derived from ethylene glycol.

Here, m is a natural number greater than 2, preferably a natural number of 3 to 100. More preferably, m is a natural number of 4 to 40.

In addition, it is preferable that the hydrophilic chain (b) and the hydrophilic linker (c) are both composed of the same hydrophilic polymer. For example, it is preferable that the hydrophilic chain (b) and the hydrophilic linker (c) both contain polyethylene glycol.

The photocleavable linker (d) contains a functional group that can be decomposed by irradiation with light such as visible light and ultraviolet light. Thus, target cells can be separated and collected from the surface of the substrate by performing light irradiation after capturing the target cells in the hydrophobic chain (a). Also, as described later, after the entire surface of the base material is modified with a photolytic cell fixing agent, the hydrophobic chain (a) is detached in advance by irradiating a specific area with light, and a desired area is obtained. It is also possible to pattern so as to have cell binding property.

The functional group of the photodegradable linker (d) that can be decomposed by light irradiation is not particularly limited as long as it can cleave the hydrophilic linker (c) and the hydrophilic chain (b) by photoreaction, -A bivalent group having a -nitrobenzyl skeleton, a coumarin-4-ylmethyl skeleton, a phenylcarbonylmethyl skeleton or a 7-nitroindolinocarbonyl skeleton can be used. Preferably, it is bivalent having a 2-nitrobenzyl skeleton.

As a specific example of the photodegradable cell fixing agent of the present invention, a compound having the following structure can be mentioned.

The compound has an oleyl group as the hydrophobic chain (a); a polyethylene glycol chain as the hydrophilic chain (b) and the hydrophilic linker (c); a 2-nitrobenzyl group in the photocleavable linker (d); And N-hydroxysuccinimide at the end of the hydrophilic chain (b) for binding to a substrate. In the formula, m is a natural number greater than 2, and n is a natural number of 50 or more. As mentioned above, m is preferably preferably a natural number of 3 to 100, more preferably m is a natural number of 4 to 8.

2. The present invention also relates to a cell immobilizing substrate having a surface modified with the above-mentioned photolyzable cell immobilizing agent. The surface structure of the cell immobilizing substrate is, as described above, a photocleavable cell fixing agent bound to the surface of the substrate directly at the end of the hydrophilic chain (b) or via a covering layer described later. is there. The photocleavable cell fixing agent is preferably arranged in the form of a monolayer on the surface of the substrate.

The material, shape, and the like of the base material to be modified by the photodegradable cell fixing agent are not particularly limited, and various suitable base materials can be selected according to the use and the like. For example, even if the shape of the substrate to be modified is a substrate (plate or film, for example, a slide glass, a dish, a microplate, a substrate for microarray, etc.), the carrier (for example, particles such as beads) or It may be colloidal), fibrous structures, tubes, containers (eg test tubes and vials). Materials of the substrate to be modified include glass; cement; ceramics or fine ceramics such as chinaware; polymer resins such as polyethylene terephthalate, cellulose acetate, polycarbonate, polystyrene and polymethyl methacrylate; biomaterials such as polypeptide and protein; silicon; Activated carbon; porous glass; porous ceramics; porous silicon; porous activated carbon; nonwoven fabric; filter paper; membrane filter; conductive material such as gold, etc. may be mentioned. In order to introduce amino groups, carboxyl groups, hydroxy groups, etc., the surface of the substrate to be modified is coated with a polymer such as polycation or treated with a silane coupling agent having a substituent introduced onto the substrate surface. It may be applied, or reactive functional groups may be introduced by plasma treatment.

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

In addition, the substrate for cell immobilization of the present invention can have a patterned surface modification having a photolytic cell fixing agent only on a specific surface area. Thereby, target cells can be immobilized on a specific area of the substrate surface. For example, in order to immobilize one cell, a plurality of spot-type modified regions can be provided. The diameter of the spot for immobilizing one cell depends on the size of the target cell, but can be approximately 2 to 30 μm, or 5 to 15 μm.

The substrate for cell immobilization having such a patterned surface modification can be prepared by the following steps as shown in FIG.
(A) modifying the entire substrate surface with a photolytic cell fixing agent, and (B) patterning the surface such that the surface modification with the photolytic cell fixing agent is maintained only in a specific region And irradiating the region other than the specific region on the substrate surface with light to cleave the photocleavable linker moiety in the photocleavable cell fixing agent.

3. Method for Immobilizing and Recovering Cells Furthermore, the present invention is also directed to a cell sorting technique in which target cells are immobilized and selectively recovered using a substrate for cell immobilization which has been surface-modified with a photolytic cell immobilization agent. Related. The cell recovery method of the present invention comprises the following steps:
(1) contacting a solution containing predetermined target cells with a substrate for cell immobilization, and immobilizing the target cells on the substrate for cell immobilization,
(2) The cell immobilization substrate is irradiated with light, and the photocleavable linker moiety in the photodegradable cell immobilization agent on the surface of the cell immobilization substrate is cut, whereby the immobilization is performed. Separating and recovering target cells from the cell immobilization substrate.

A base for cell immobilization can be placed in the microchannel as a place to perform the method. In step (2), a flux can be applied to the surface of the substrate to recover target cells after cleavage of the photocleavable linker moiety.

As described above, it is possible to immobilize and recover one cell by patterning the substrate surface and having a plurality of spot-type modified regions for immobilizing one cell.

Hereinafter, the present invention will be described in more detail by way of examples, but the present invention is not limited thereto.

1. Synthesis of Photodegradable Cell Fixing Agent Synthesis of PEG lipid (PEG ₄ ) P-BAM, which is a photodegradable cell fixing agent of the present invention, was performed according to the following scheme. (PEG ₄ ) P-BAM has a hydrophilic linker of m = 4. The synthesis was performed in the same manner for m = 2, 8 and 40 different in the length of the hydrophilic linker moiety.

100mlナス型フラスコに化合物1 (2.9950 g, 18.0 mmol)、K₂CO₃ (4.0808 g)、DMF (20 ml)、4-ブロモ酪酸メチル (2.6 ml, 20.6 mmol, 1.14 eq)を順に加え撹拌。o/nで撹拌後TLC (CHCl₃:CH₃OH=10:1, Rf: compound 1 = 0.70, target=0.75)で原料(化合物1)の消失を確認。純水をK₂CO₃やKBrが溶けるまで加え、TLCで確認しながら酢酸エチルとBrineで分液を三回行い、水層から生成物を回収した。有機層を三角フラスコに回収して硫酸マグネシウムで乾燥後、吸引ろ過で硫酸マグネシウムを除去し、溶媒を減圧除去して薄い褐色のオイルを得た。未反応の4-ブロモ酪酸エチルを除くため、シリカカラム精製 (酢酸エチル:ヘキサン=4:6)を行った。目的の生成物を21-44本目に回収し(49本中)、減圧留去後、o/nで真空乾燥し化合物2を得た。同定は¹H-NMR (CDCl₃)で行った(Fig. 5-7)。収量は4.2434 g、収率は88 %であった 1-1. Synthesis of compound 2

100ml eggplant type flask Compound 1 (2.9950 g, 18.0 mmol) , K 2 CO 3 (4.0808 g), DMF (20 ml), 4- bromo-butyric acid methyl (2.6 ml, 20.6 mmol, 1.14 eq) and stirring was added sequentially. After stirring at o / n, disappearance of the raw material (compound 1) was confirmed by TLC (CHCl ₃ : CH ₃ OH = 10: 1, Rf: compound 1 = 0.70, target = 0.75). Pure water was added until K ₂ CO ₃ and KBr were dissolved, and the solution was separated three times with ethyl acetate and Brine while confirming by TLC, and the product was recovered from the aqueous layer. The organic layer was collected in an Erlenmeyer flask and dried over magnesium sulfate, and then the magnesium sulfate was removed by suction filtration, and the solvent was removed under reduced pressure to obtain a pale brown oil. In order to remove unreacted ethyl 4-bromobutyrate, silica column purification (ethyl acetate: hexane = 4: 6) was performed. The desired product was collected at the 21st to 44th columns (of 49), evaporated under reduced pressure, and dried in vacuo at o / n to give compound 2. Identification was performed by ¹ H-NMR (CDCl ₃ ) (Fig. 5-7). The yield was 4.2434 g, 88%

実験は全てドラフト内で行った。on iceで200 mlナスフラスコに69 %硝酸(60 ml)をいれ、そこに撹拌しながら無水酢酸(12 ml)をパスツールピペットで少しずつ滴下した。さらに無水酢酸(12 ml)に溶かした化合物2 (3.0021 g, 11.2 mmol)をパスツールピペットで少しずつ滴下したところ、溶液が黄色透明になった。2 h 30 min撹拌した後、on ice (with NaCl)で冷やしておいた三角フラスコ中の純水(300 ml)にパスツールピペットで反応液を少しずつ滴下し、薄黄色沈殿を得た。(間違って-30 °C で一晩静置した後)、4℃で5 h静置し、沈殿を吸引濾過で回収して純粋で洗浄後、デシケーターで一晩真空乾燥させ、薄黄色の化合物3を得た、収量は1.8726 g。収率は53.4 %であった。同定は¹H-NMR(CDCl₃)でおこなった(Fig. 5-8)。 1-2. Synthesis of compound 3

All experiments were conducted in the draft. 69% nitric acid (60 ml) was put into a 200 ml eggplant flask on ice, and acetic anhydride (12 ml) was dropped little by little with a Pasteur pipette while stirring there. Furthermore, when the compound 2 (3.0021 g, 11.2 mmol) dissolved in acetic anhydride (12 ml) was dripped little by little with a Pasteur pipette, the solution became yellowish transparent. After stirring for 2 h 30 min, the reaction solution was added little by little with a Pasteur pipette to pure water (300 ml) in an Erlenmeyer flask cooled with on ice (with NaCl) to obtain a pale yellow precipitate. (Incorrectly after standing at -30 ° C overnight), stand at 4 ° C for 5 h, collect precipitate by suction filtration, wash with pure, vacuum dry with a desiccator overnight, light yellow compound Obtained 3, the yield is 1.8726 g. The yield was 53.4%. Identification was performed by ¹ H-NMR (CDCl ₃ ) (FIG. 5-8).

実験は全てドラフト内で行った。100 ml枝付きナスフラスコ内に化合物3 (1.4953 g, 4.8 nmol)を入れ、dry up、N₂置換後にdry THF (25 ml)とdry MeOH (14 ml)を加えon iceで撹拌したところ黄色に懸濁した溶液になった。NaBH₄ (0.3323 g, 8.8 mmol, 1.83eq)を粉末として加え、気体が発生している間はon iceで撹拌し、気体が収まった後はr.t.でo/nで反応させた(頂いた資料では15 h反応)。TLC (CHCl₃:CH₃OH=20:1, Rf:原料=0.55, 反応中間体=0.38)で原料消失を確認後、4 M NaOH (3 ml)と純水(10 ml)を加えて次の加水分解の反応に移った。3 h 30 min反応後、TLC (Rf：target=0.14)で反応中間体消失ｍを確認し、溶媒を減圧除去。黄褐色透明溶液になったが、その後を引き続けたところ褐色固体になった。ここに10 M HCl (2 ml)と純水を加えたところ溶液がコロイド化してしまった。約200 mlの酢エチを加えて溶解させた後、酢エチで抽出。TLCで水層に生成物が無いことを確認後、MgSO₄を加えて乾燥し、吸引濾過でMgSO₄を除去した後、溶媒を減圧除去した。真空乾燥を経てオレンジ色の固体を得た。その後、生成物を酢エチに溶かして減圧除去で飽和させ、ヘキサンを滴下時の靄が消えなくなるまで滴下し、-20℃で静置、析出した沈殿を吸引濾過で回収することを3回繰り返し、再結晶をおこなった。この際1,2回目は黄白色のペースト状の固体、3回目はオレンジ色の粉末が得られた。得られた固体を真空乾燥し、化合物4を得た。同定は¹H-NMR (DMSO-d6)で行い(Fig. 5-9)、収量は1.0992 g (1,2回目0.95 g、3回目0.1492 g)、収率は76.4 %であった。 1-3. Synthesis of compound 4

All experiments were conducted in the draft. Compound 3 (1.4953 g, 4.8 nmol) was placed in a 100 ml branched eggplant flask, and after dry up, N ₂ substitution, dry THF (25 ml) and dry MeOH (14 ml) were added and stirred with ice. It became a suspended solution. NaBH ₄ (0.3323 g, 8.8 mmol, 1.83 eq) was added as a powder, and while gas was being generated, stirring was performed on ice, and after the gas was settled, reaction was performed at o / n at rt (data provided) In the 15 h reaction). After confirming the disappearance of the raw materials by TLC (CHCl ₃ : CH ₃ OH = 20: 1, Rf: raw material = 0.55, reaction intermediate = 0.38), 4 M NaOH (3 ml) and pure water (10 ml) are added, and the next Transferred to the reaction of hydrolysis of After 3 h 30 min reaction, disappearance of reaction intermediate m is confirmed by TLC (Rf: target = 0.14), and the solvent is removed under reduced pressure. It became a yellow-brown clear solution, but it continued to become a brown solid as it continued to pull. When 10 M HCl (2 ml) and pure water were added to the solution, the solution became colloidal. Add about 200 ml of vinegar and dissolve it, then extract with vinegar. After confirming that there was no product in the aqueous layer by TLC, the reaction solution was added with MgSO ₄ for drying, and after suction filtration to remove MgSO ₄ , the solvent was removed under reduced pressure. After vacuum drying, an orange solid was obtained. Thereafter, the product is dissolved in vinegar and saturated by removal under reduced pressure, hexane is added dropwise until the drop in the dropping does not disappear, and the mixture is allowed to stand at -20 ° C, and the precipitated precipitate is collected by suction filtration three times repeatedly. , Recrystallized. At this time, a yellowish white paste-like solid was obtained in the first and second times, and an orange powder was obtained in the third time. The resulting solid was dried under vacuum to give compound 4. The identification was performed by ¹ H-NMR (DMSO-d6) (FIG. 5-9), and the yield was 1.0992 g (1,2nd 0.95 g, 3rd 0.1492 g), and the yield was 76.4%.

50 mlふた口フラスコにEDC 442.6 mg (2.30 mmol)とHOBt 154.9 mg (1.14 mmol)を加えて真空引き、窒素置換後20 mlのdry DCMを加えて撹拌し完全に溶解させた。ここに387.6 mg (1.06 mmol)の化合物9をテルモシリンジで加えた後、450 μl (1.36 mmol)のOleylamineをテルモシリンジで加えた。学会を挟んで約6日撹拌後TLC (DCM/MeOH=7/1, Rf: 化合物9=0.5, oleylamine=0.16, product=0.58)で原料の消失を確認し、シリカカラム(DCM/MeOH=9/1)にかけ、目的物画分を濃縮し淡黄色のオイルを得た。収量は572.6 mg、収率は84.5 %だった。¹H-NMR (DMSO-d6)とESI-MSにより目的物の生成を確認した(Fig. 5-10)。HOBtが0.17当量混入したものが得られた。 1-4. Synthesis of compound 10

442.6 mg (2.30 mmol) of EDC and 154.9 mg (1.14 mmol) of HOBt were added to a 50 ml two-necked flask, vacuum was applied, 20 ml of dry DCM was added after nitrogen substitution, and the mixture was stirred to dissolve completely. After 387.6 mg (1.06 mmol) of Compound 9 was added thereto via a thermo syringe, 450 μl (1.36 mmol) of Oleylamine was added via a thermo syringe. After stirring for about 6 days across the academic society, disappearance of the raw materials was confirmed by TLC (DCM / MeOH = 7/1, Rf: compound 9 = 0.5, oleylamine = 0.16, product = 0.58), and a silica column (DCM / MeOH = 9) The desired product fraction was concentrated to give a pale yellow oil. The yield was 572.6 mg, and the yield was 84.5%. The formation of the desired product was confirmed by ¹ H-NMR (DMSO-d6) and ESI-MS (FIG. 5-10). What HOBt mixed with 0.17 equivalent was obtained.

1-5. Synthesis of Compound 12

1st process
(in the case of m = 4)
100.7 mg (163 μmol) of the compound 10 was put in a 20 ml two-necked eggplant flask, 3 ml ethyl acetate was added and stirred, and 1.5 ml 12 M HCl was dropped thereto. After completion of the reaction at o / n, TLC (DCM / MeOH = 7/1, Rf: 10 = 0.67, 11 = 0.23, iodine color) confirmed completion of the reaction, the solvent was removed by an evaporator, and the residue was dried under vacuum.

(in the case of m = 8) * also in the case of m = 2
39.6 mg (43.8 μmol) of compound 10 was added to a 50 ml eggplant flask and dissolved in 5 ml of MeOH. 0.5 ml of 2 M NaOH aq was added there, and it stirred at room temperature for 1 hour. The termination of the reaction was confirmed by TLC (DCM / MeOH = 10/1, Rf: 10 = 0.49, 11 = 0.1). After MeOH was distilled off, extraction was performed three times with ethyl acetate, dried over sodium sulfate, and concentrated and vacuum dried with an evaporator.

2nd process
(in the case of m = 4) * 65.4 mg (218 μmol) of the compound 4 and 63.6 mg (331 μmol) of EDC with a two-necked eggplant flask containing the crude compound 11 also in the case of m = 2, 8 After adding 200 μl dry TEA and evacuating and replacing with nitrogen, the solution was stirred with dry DCM to obtain a yellow clear solution. After completion of the reaction at o / n, TLC confirmed completion of the reaction, applied to silica column (DCM / MeOH = 7/1), target fraction was confirmed by ESI, concentrated and dried under vacuum to obtain brown oil The The yield is 138 mg and the yield is 76.4% taking into account by-products.

50 ml二口ナスフラスコに化合物12を46.6 mg (60.8 μmol)とクロロギ酸ニトロフェニルを10.9 mg (54.2 μmol)加えて一晩真空乾燥後、窒素置換し10 mlのdry DCMと、200 μlのdry TEAを加えて反応を開始させた。約二日撹拌してもTLC (DCM/MeOH=8/1, Rf: s.m.=0.53, target=0.60)で反応が進行している様子が確認できず、ESI、¹H-NMR (DMSO-d6)でも反応の進行が確認できなかったため、溶媒を除去し、70 mg (348 μmol)のクロロギ酸ニトロフェニルを加えて真空乾燥、窒素置換後、dry DCMを10 mlとdry TEAを100 μl加えて反応を開始させた。2時間後、TLCで反応の終了を確認し、溶媒を除去、真空乾燥後、シリカカラム(DCM/MeOH=10/1)にかけたところ、目的物とクロロギ酸ニトロフェニルが分離できなかったため、シリカカラム(DCM/MeOH=20/1)にかけなおし、目的物画分をESIで確認、濃縮した。収量は85.2 mg、収率は85 %であった。クロロギ酸ニトロフェニルの加水分解体が3.29等量含まれているものが得られた。同定は¹H-NMR (DMSO-d6)およびESI-MSにより行った(Fig. 5-12)。 1-6. Synthesis of compound 13

After adding 46.6 mg (60.8 μmol) of compound 12 and 10.9 mg (54.2 μmol) of nitrophenyl chloroformate to a 50 ml two-necked eggplant flask and drying under vacuum overnight, the flask is purged with nitrogen and 10 ml of dry DCM and 200 μl of dry The reaction was initiated by the addition of TEA. Even after stirring for about two days, it was not possible to confirm that the reaction was proceeding by TLC (DCM / MeOH = 8/1, Rf: sm = 0.53, target = 0.60), and ESI, ¹ H-NMR (DMSO-d6 But the reaction progress could not be confirmed, so remove the solvent, add 70 mg (348 μmol) of nitrophenyl chloroformate, vacuum dry, replace with nitrogen, add 10 ml of dry DCM and 100 μl of dry TEA. The reaction was started. After 2 hours, TLC confirmed the completion of the reaction, the solvent was removed, the residue was dried under vacuum and then applied to a silica column (DCM / MeOH = 10/1), the target substance and nitrophenyl chloroformate could not be separated, so silica The column (DCM / MeOH = 20/1) was applied again, and the objective fraction was confirmed by ESI and concentrated. The yield was 85.2 mg, and the yield was 85%. One containing 3.29 equivalents of the hydrolyzate of nitrophenyl chloroformate was obtained. Identification was performed by ¹ H-NMR (DMSO-d6) and ESI-MS (FIG. 5-12).

50 ml二口ナスフラスコに保存していた65.3 mg (40.13 μmol)の化合物13に対し、101.4 mg (29.8 μmol)のPA-034HCを加え、dry up、窒素置換後、10 mlのdry DCMと200 μlのdry TEAを加えて反応を開始させた。約二日撹拌後、TLC(DCM/MeOH=6/1, Rf: 13=0.48, PA-034HC=0.33, target=0.44?) で反応の終了を確認し、エーテル沈殿で目的物を回収、混入していたジクロロメタンに不要な固形物を濾過で除いて目的物を得た。この時点での収量は125.1 mg、収率は99 %であった。¹H-NMR (DMSO)を取ったところクロロギ酸ニトロフェニルの加水分解体が混入していたため、試しにエタノール沈殿にかけてみた後に¹H-NMR (DMSO)を取ったところ、純度良く目的物が得られた。最終的な収量は89 mg、収率は70.6 %であった。PA-034HCの未反応物のピークはNMRで観察されなかった。同定は¹H-NMR (DMSO-d6)により行った(Fig. 5-13)。 1-7. Synthesis of Compound 14

To 65.3 mg (40.13 μmol) of compound 13 stored in a 50 ml two-necked eggplant flask, 101.4 mg (29.8 μmol) of PA-034HC was added, and dry up, after nitrogen substitution, 10 ml of dry DCM and 200 The reaction was initiated by adding μl of dry TEA. After stirring for about two days, the end of the reaction is confirmed by TLC (DCM / MeOH = 6/1, Rf: 13 = 0.48, PA-034HC = 0.33, target = 0.44?), And the target substance is recovered by ether precipitation and contaminated The unnecessary solid was removed by filtration to obtain the desired product. The yield at this point was 125.1 mg, and the yield was 99%. ^{When 1} H-NMR (DMSO) was taken, the hydrolyzate of nitrophenyl chloroformate was mixed. Therefore, after trying ethanol precipitation as a trial, ¹ H-NMR (DMSO) was taken, and the desired product was obtained with high purity. It was done. The final yield was 89 mg, and the yield was 70.6%. The peak of unreacted PA-034 HC was not observed by NMR. Identification was performed by ¹ H-NMR (DMSO-d6) (Fig. 5-13).

50 mlの二口ナスフラスコに55.5 mg (13.1 μmol)の化合物14と146.6 mg (711 μmol)のDCC、16.4 mg (142 μmol) のNHSを加え、真空乾燥、窒素置換後、10 mlのdry DCMと100 μlのdry TEAを加えて反応を開始させた。約二日撹拌後、反応液を濾過、濃縮後、エーテル沈殿で目的物を回収し、¹H-NMR (DMSO)で同定を行った(Fig. 5-14)。DCCのウレアが0.38 eq含まれたものが得られ、収量は53.5 mg、収率は92 %だった。実際の分子量は4404として今後も扱った。(目的物のMw=4319) 1-8. Synthesis of Compound 15 ((PEG ₄ ) P-BAM)

Add 55.5 mg (13.1 μmol) of compound 14 and 146.6 mg (711 μmol) of DCC, 16.4 mg (142 μmol) of NHS to a 50 ml two-necked eggplant flask, vacuum dry, replace with nitrogen, and then 10 ml of dry DCM The reaction was started by adding 100 μl of dry TEA. After stirring for about two days, the reaction solution was filtered and concentrated, and then the target substance was collected by ether precipitation, and identification was performed by ¹ H-NMR (DMSO) (FIG. 5-14). One containing 0.38 eq of urea of DCC was obtained, the yield was 53.5 mg, and the yield was 92%. The actual molecular weight was further treated as 4404. (Object Mw = 4319)

2. Substrate preparation Surface modification of the glass substrate with the photodegradable cell fixing agent was performed in the following procedure.

2-1. Initial Cleaning of Substrate The slide glass was placed in a dose and subjected to an ultrasonic cleaner for 10 minutes in water containing alkaline scat. The same operation was carried out by replacing water containing alkaline scat with acetone, and after performing the same washing in the order of isopropanol → acetone → isopropanol, air-dried.

2-2. Collagen coating on the base A spacer was placed at each end of each well of the 4-well dish so that the slide glass did not touch the bottom of the well. Collagen (3 mg / ml, pH 3) was diluted 10-fold with MQ adjusted to pH 3 with HCl to make 0.3 mg / ml, and 4 ml was poured into each well of 4-well dishes. The slide glass which had been subjected to the initial washing was immersed one by one in each well, shielded from light and allowed to stand overnight at room temperature. The next day, after rinsing three times with MQ using a dose, air-dried. Store used spacers in ethanol for reuse. When coating a 35 mm glass bottom dish, the solution volume was 200 μl and washing was performed using an aspirator or pipetman.

2-3. Photolytic cell immobilization agent (PEG lipid) modification of substrate (spot method)
The PEG lipid to be used for modification was prepared by dissolving it in dry DMSO so as to be 10 mM, aliquoted in 5 μl aliquots, and stored at −20 ° C. after Ar substitution. This stock solution is diluted with 495 μl of PBS- to 100 μM immediately before use, and placed in a 4-well dish where 0.3 μl of total 0.3 μl spots are placed at 1 cm intervals in the center of the channel, and the incubator (37 ° C, Incubate for 1 hour in 5% CO ₂ ), then wash 6 times with MQ and air dry.

3. Cell Regulation 3-1. Thaw / culture / freeze cells
The thawed serum-containing medium was warmed in a thermostat at 37 ° C., and the frozen cells were thawed in the thermostat until some ice remained. Immediately after complete dissolution, the whole was transferred to a 15 ml tube and diluted with 9 ml of medium. The supernatant was removed by centrifugation (190 G, 3 min, same or less), resuspended in 10 ml of medium and plated in a 100 mm dish.
Cultivation Incubate basically in an incubator (37 ° C, 5% CO ₂ ). The medium composition is as follows.
eGFP expressing Ba / F3: RPMI (10% FBS), 1 ng / ml IL3
Passage Ba / F3 is only diluted in basic fresh medium.
Frozen cells are collected in a 15 ml tube, washed once with a clean medium, suspended in Cell Banker, aliquoted in 0.5 or 1 ml aliquots, and stored at -80 ° C. Long term storage samples were stored in liquid nitrogen.

3-2. Pretreatment of cells for cell fixation After collecting Ba / F3 by centrifugation (100 G, 3 min, same or less), wash twice with 10 ml PBS- to 3 × 10 ⁶ cells / ml Were resuspended in PBS- as indicated.

4. Photopatterning Photoirradiation was used with Asahi Spectrometer MAX-302, and a filter was used with LX0360 (360 nm ± 2 nm). The light was automatically emitted using the MAX-302 timer function based on the amount of light measured by the light amount meter. When irradiating through a photo mask, the surface of the mask of the photo mask faces up and the base of the substrate is modified with the PEG-modified surface facing down, with respect to light from below. Contact exposure was performed by putting.

5. Evaluation of Cell Fixation Ability Next, the cell fixation ability was evaluated using a substrate modified with a photolytic cell fixation agent (PEG lipid).

5-1. Creation of channel system The parts of the channel including the tube were combined to create a microchannel. When connecting the flow paths, care was taken not to apply pressure and to prevent air bubbles from being mixed. Ibidi's cover-type flow path is coated by soaking in 1% BSA for 10 minutes in 4-well dishes at the first use, and after use, ultrasonic cleaning is performed using alkali scat-added water and use went.

5-2. Evaluation of cell immobilization power of PEG lipid Various PEG lipids are modified in the microchannel and immobilized on the bottom of the channel at a density at which cells do not contact each other. Buffer was flowed into the channel at various flow rates, and the percentage of cells remaining before and after that was counted. As the PEG lipid, the photolyzable cell immobilization agent according to the present invention synthesized in the above 1 was used. (PEG2) P-BAM, (PEG4) P-BAM, (PEG8) P-BAM, (PEG40) P, wherein the length of the hydrophilic linker is m = ₂ , ₄ , ₈ , _{40, respectively} Four types of-BAM were used.

The same experiment was also performed on a photocleavable PEG lipid having no hydrophilic linker as a comparative example. The PEG lipid of the comparative example has the following structure and has a photocleavable linker between the hydrophobic chain and the hydrophilic chain, but the hydrophobic chain and the photocleavable linker are directly linked: It does not have a hydrophilic linker.

The results are shown in FIG. As a result, in the conventional PEG-free photodegradable PEG lipid, 80% or more of the cells were detached without being able to withstand shear stress, but a photo-degradable PEG lipid having m = 4 to 40 hydrophilic linkers At least 80% of the cells remained. Thus, it was shown that incorporation of a hydrophilic linker between a hydrophobic chain and a photocleavable linker can significantly improve the cell immobilization power.

5-3. Cell detachment by light irradiation Next, cells are immobilized so as to isolate cells on the surface modified with a photocleavable PEG lipid ((PEG 8) P-BAM) having a hydrophilic linker of m = 8. The cell-immobilized surface is rinsed at a flow rate of 3 ml / min after being irradiated with light of a high intensity (365 nm), and the cell survival rate decreases according to the amount of light irradiation, and approximately 3 J / cm ² of light All cells were removed. The results are shown in FIG. Thus, by using the photodegradable cell fixing agent of the present invention, in which the cell immobilization power is enhanced by incorporating a hydrophilic linker, it is possible to selectively recover the cells immobilized one by one by light irradiation. Indicated.

5-4. Preparation and collection of cell array According to the above 4, the bottom surface of the microchannel is irradiated with a pattern of ultraviolet light (365 nm) of 3 J / cm ² or more, and the surface on which cells are not immobilized The region where the hydrophobic chain part of the degradable cell fixing agent was released and the surface (non-irradiated region) to be fixed were patterned. The cell suspension is introduced into the microchannel, and the cells are immobilized through the interaction of the hydrophobic chain of the photolytic cell fixing agent in the non-irradiated area with the cells, and the cells are about 2 to 5 ml / min. The cells non-specifically adsorbed to the light-irradiated area are washed out by rinsing by adjusting the flow rate to become Thereby, a cell array was produced in the microchannel.

FIG. 6 shows an image of the obtained 1-cell microarray. FIG. 6 (a) is a fluorescence image of an EGFP-expressing BaF3 cell array formed on a cell immobilization substrate coated with collagen as a covering layer. FIG. 6 (a) is a bright field image of an EGFP-expressing BaF3 cell array formed on a cell immobilization substrate coated with BSA as a coating layer.

Next, selective detachment in one cell unit was performed on the obtained cell array. The flow rate was adjusted to be about 0.5 ml / min, and only the target cells were irradiated with visible light (405 nm) to desorb the cells. Fluorescence images before and after selective desorption are shown in FIG. As shown in FIG. 7, when light was irradiated only in the frame of the left figure, it was confirmed that only the cells present in the frame were selectively detached.

The cell detachment rate in the light-irradiated spot and the non-irradiated spot calculated by analysis of the fluorescence image before and after light irradiation is shown in FIG. This result shows that cells can be selectively detached by light irradiation by using the substrate for cell immobilization of the present invention.

5-5. Differentiation to dendritic cells on cell array
A 1-cell array was formed on a substrate for cell immobilization (collagen coating) in the same manner as in 5-4 above, and monocytes collected from mice were cultured for 6 days. An image (superimposed image of fluorescence and bright field) after immunostaining with an anti-CD11 antibody PE conjugate is shown in FIG. The formation of dendritic cells was confirmed, and it was confirmed that initial culture / differentiation on the cell array surface is possible. The differentiation efficiency was 45%.

Claims (16)

A photolytic cell immobilization agent for immobilizing predetermined target cells on a substrate, comprising:
A hydrophobic chain capable of interacting with the target cell,
It has a hydrophilic chain that can be arranged in the form of a monolayer on the surface of the substrate, and a hydrophilic linker and a photocleavable linker that connect the hydrophobic chain and the hydrophilic chain,
The photocleavable cell fixing agent, wherein the hydrophilic linker is disposed between the hydrophobic chain and the photocleavable linker. The photodegradable cell fixing agent according to claim 1, wherein the hydrophilic linker comprises a hydrophilic polymer. The photodegradable cell fixing agent according to claim 1, wherein the hydrophilic linker comprises a polyalkylene glycol. The photocleavable cell fixing agent according to claim 1, wherein the hydrophilic linker has the following partial structure.

(Wherein, m is a natural number greater than 2) The photodegradable cell fixing agent according to claim 4, wherein m is a natural number of 4 to 40. The photodegradable cell fixing agent according to any one of claims 1 to 5, wherein the hydrophobic chain is a saturated or unsaturated hydrocarbon chain which may have a substituent. The photodegradable cell fixing agent according to any one of claims 1 to 6, wherein the hydrophilic chain comprises a hydrophilic polymer. The photodegradable cell fixing agent according to any one of claims 1 to 6, wherein the hydrophilic chain comprises a polyalkylene glycol. The photolyzable linker is a divalent group having a 2-nitrobenzyl skeleton, a coumarin-4-ylmethyl skeleton, a phenylcarbonylmethyl skeleton or a 7-nitroindolinoxycarbonyl skeleton. Photodegradable cell fixing agent as described. The photocleavable cell fixing agent according to any one of claims 1 to 9, wherein the hydrophilic chain and the hydrophilic linker both include polyethylene glycol. The photodegradable cell fixing agent according to any one of claims 1 to 10, which has a substituent which can be covalently bonded to the surface of the substrate at the end of the hydrophilic chain. The photodegradable cell fixing agent according to claim 1, which has the following structure.

(Wherein, m is a natural number of 2 to 40, and n is a natural number of 45 to 500) A substrate for cell immobilization, which has a surface modified by the photolytic cell immobilization agent according to any one of claims 1 to 12. The cell immobilization substrate according to claim 13, having a patterned surface modification having a photolytic cell immobilization agent only on a specific surface area of the substrate. A step of modifying the entire substrate surface with the photolytic cell fixing agent according to any one of claims 1 to 12, and a surface such that the surface modification with the photolytic cell fixing agent is maintained only in a specific region. Patterning the substrate surface by irradiating light to a region other than the specific region on the substrate surface, and cleaving the photodegradable linker moiety in the photodegradable cell fixing agent.
A method for producing a substrate for cell immobilization, comprising Allowing a solution containing a predetermined target cell to contact the cell immobilization substrate according to claim 13 or 14, and immobilizing the target cell on the cell immobilization substrate.
The immobilized target cells are obtained by irradiating the substrate for cell immobilization with light and cleaving the photodegradable linker moiety in the photolytic cell immobilization agent on the surface of the substrate for cell immobilization. Separating and recovering from the substrate for cell immobilization,
Cell recovery methods, including:

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