WO2017163463A1

WO2017163463A1 - Cell sorting method and flow cytometry and cell sorter using same

Info

Publication number: WO2017163463A1
Application number: PCT/JP2016/077645
Authority: WO
Inventors: 哲志山口; 岡本　晃充; 理沙高木; 博行野地; 亮太飯野; 和仁田端
Original assignee: 国立研究開発法人科学技術振興機構
Priority date: 2016-03-25
Filing date: 2016-09-20
Publication date: 2017-09-28
Also published as: JP2019090610A

Abstract

[Problem] To provide an automatable cell acquisition technique by which a plurality of cells can be simultaneously analyzed while reducing the frequencies of false positives and false negatives. [Solution] A cell sorter or flow cytometer comprising a flow passage provided with a plurality of microwells, an introduction passage capable of introducing a liquid, said liquid containing a plurality of target cells, into the flow passage, a data acquisition part acquiring data from the target cells housed in the microwells, a selective taking-out means capable of selectively taking out the target cells, said target cells being in the microwells, from the microwells on the basis of the data acquired by the data acquisition part, and a target cell collection part capable of collecting the target cells selectively taken out by the selective taking-out means, characterized in that: the microwells can house the target cells and a photodegradable gel; and the selective taking-out means is a degrading light-irradiation part capable of irradiating the photodegradable gel, said photodegradable gel being in a microwell housing a target cell to be taken out, with light having a wavelength at which the photodegradable gel degrades.

Description

Cell sorting method, flow cytometry and cell sorter using the same

The present invention relates to a novel cell sorting method, and a flow cytometry and cell sorter using the same.

Flow cytometry is a technique in which antibodies labeled with a fluorescent dye are bound to target cells, and particles are analyzed and separated by the fluorescence and scattered light. At this time, a technique for separating specific cells (including microorganisms) from a population of fine particles such as cells is a cell sorting technique (Non-patent Document 1). Cell sorting technology is a very important method for understanding cell behavior and cell function.

In the technique of Non-Patent Document 1 (see FIG. 18), a suspension of target cells to which an antibody labeled with a fluorescent dye or the like is bound is used as a liquid flow. In response to the excitation light, the desired cells are identified by analyzing the wavelength or intensity of the fluorescence or scattered light emitted from each cell. Next, voltage is applied to the cells having specific properties identified by the analysis results such as the wavelength and intensity of the light, and the cells are charged by using a deflection electrode to discriminate, quantify and statistically analyze the charged cells. Etc.

Prior literature

As described above, the conventional cell sorting technique is a technique for sequentially analyzing and recovering each cell flowing along a flow path. More specifically, the analysis is based on the absolute value of a signal (mainly fluorescence or scattered light) emitted from a cell at the moment of passing through the detection unit. Therefore, it is not suitable for analyzing many items for many cells. Moreover, this technique is also a technique that is concerned about the occurrence of false positives and false negatives. On the other hand, as a technique for reducing the frequency of false positives and false negatives and analyzing many items for many cells, there is a microarray technique. However, in this technique, when a cell desired to be acquired is found, the cell is acquired by a technique of inserting a micromanipulator into a microwell containing the cell. Is unsuitable. Therefore, it is an object of the present invention to provide a subject (eg, cell) acquisition technique that can simultaneously analyze a plurality of subjects (eg, cells) while reducing the frequency of false positives and false negatives and that can be automated. And

The present invention is as follows:
[1] a flow path having a plurality of microwells;
An introduction path capable of introducing a liquid containing a plurality of specimens (for example, test cells) into the flow path;
An information acquisition unit for acquiring information from a plurality of specimens (for example, test cells) stored in the plurality of microwells;
Based on the information acquired by the information acquisition unit, a selective extraction means capable of selectively extracting a specimen (for example, a test cell) in one microwell from the microwell;
A cell sorter or flow cytometer having a specimen (eg, test cell) collection unit capable of collecting the specimen (eg, test cell) selectively taken out by the selective extraction means,
The plurality of microwells can store a specimen (for example, a test cell) and a degradable gel (for example, a photodegradable gel),
When the selective extraction means has a degradable gel (for example, photodegradable gel) in a microwell in which a specimen (for example, a test cell) to be extracted is held, the degradable gel (for example, photodegradable gel) A cell sorter or flow cytometer configured to be a decomposition processing unit (for example, a decomposition light irradiation unit) that performs a decomposition process (for example, can irradiate light having a wavelength at which the photodegradable gel decomposes).
[2] The cell sorter according to [1], wherein the flow channel is planar, and the plurality of microwells are provided on a lower wall (of the upper wall and the lower wall) constituting the flow channel. Flow cytometer.
[3] The degradable gel is a photodegradable gel,
The cell sorter or flow cytometer according to [1] or [2], wherein the decomposition processing unit is a light irradiation unit capable of irradiating light having a wavelength at which the photodegradable gel is decomposed.
[4] The information acquisition unit includes:
The information from the plurality of microwells can be acquired for each microwell, two or more of the plurality of microwells can be acquired at once, or all the plurality of microwells can be acquired at once. 1] to [3] cell sorter or flow cytometer.
[5] The information acquisition unit includes:
An information source wave irradiation unit capable of irradiating the plurality of microwells with an information source wave serving as a source of the information to be acquired;
The information source wave irradiating unit includes an information receiving unit configured to receive the information that can be generated from the plurality of microwells when the information source wave is irradiated to the plurality of microwells. [4] Cell sorter or flow cytometer.
[6] Whether the information source wave irradiation unit can irradiate the information source wave for each microwell as a first aspect; as a second aspect, two or more of a plurality of microwells at a time Or, as a third embodiment, all of the plurality of microwells can be irradiated at once; in the case of the first embodiment and the second embodiment, all of the plurality of microwells are scanned. The cell sorter or flow cytometer according to [5] above, which can be irradiated.
[7] The cell sorter or flow cytometer according to [5] or [6], wherein the information source wave irradiation unit is capable of outputting a plurality of types of information source waves according to the number of analysis items.
[8] The information source wave irradiated from the information source wave irradiation unit is light, the degradable gel is a photodegradable gel, and the decomposition processing unit decomposes the photodegradable gel. The cell sorter or flow cytometer according to [5] to [7], wherein when the light irradiation unit is capable of irradiating light of a wavelength, the wavelength of the light is different from the wavelength of the light decomposed by the photodegradable gel.
[9] The light irradiation unit and the information source wave irradiation unit are the same light source,
The same light source can switch and irradiate light having a wavelength at which the photodegradable gel decomposes and light having a wavelength different from the wavelength at which the photolytic gel is decomposed, which is a source of the information to be acquired. The cell sorter or flow cytometer according to [8], which is configured as described above.
[10] The cell sorter or flow cytometer is:
It may have an automatic analysis means for automatically analyzing the information acquired by the information acquisition unit,
Furthermore, the gel is applied to a degradable gel (for example, a photodegradable gel) in a microwell containing a sample (for example, a test cell) desired to be obtained based on a user instruction or an analysis result by the automatic analysis means. The cell sorter or flow according to any one of [1] to [9], further comprising selective extraction control means for controlling the selective extraction means so as to perform a decomposition process (for example, irradiation with light having a wavelength at which the photodegradable gel decomposes). Cytometer.
[11] A microfluidic device including the flow path, which is for the cell sorter or the flow cytometer according to the above [1] to [10].
[12] A liquid containing a material capable of forming the degradable gel, which is used for the cell sorter or flow cytometer according to [1] to [10] (for example, a liquid containing the following first component; A liquid containing at least a component-containing liquid).
[13] A filling step of flowing a liquid containing a specimen (for example, a test cell) into a flow path having a plurality of microwells, and filling the specimen (for example, the test cell) into the microwell;
After the filling step, an analysis step for analyzing the specimen (for example, a test cell) filled in the microwell;
After the analysis step, a subject (eg cell) recovery method comprising an acquisition step of taking out the subject (eg test cell),
The liquid contains a first component and a second component different from the first component,
The first component has a first portion in which the basic skeleton is a biocompatible polymer and can be bonded to the second component;
The second component has a second part in which the basic skeleton is a biocompatible polymer and can bind to the first component;
In the analysis step, a gel in which the first component and the second component are cross-linked is formed in the microwell by the bond formation between the first part and the second part. ,
The first component and / or the second component further has a degradable portion (for example, a photodegradable portion),
In the acquisition step, decomposition processing (for example, light irradiation) is performed to decompose the decomposable portion (for example, photodegradable portion) to decompose the gel and take out the specimen (for example, test cell). A method for recovering a subject (eg, a cell).
[14] The subject (eg, cell) recovery method of [13], wherein the first part is an azide and the second part is an alkyne.

According to the present invention, it is possible to provide a subject (eg, cell) acquisition technique that can simultaneously analyze a plurality of subjects (eg, cells) while reducing the frequency of false positives and false negatives and that can be automated. .

FIG. 1 is a cross-sectional view of the microfluidic device A. FIG. 2 is a top view of the microfluidic device A. FIG. FIG. 3 is an exploded view of the microfluidic device A. FIG. FIG. 4 shows an example of a chemical structural formula of a bonding portion at the time of gelation between the first component and the second component that are gelling components, and a decomposition portion when light having a wavelength at which the gel decomposes is irradiated. It is an example of the chemical structural formula. FIG. 5 is a diagram showing a state in which a test cell is fixed in the microwell by using the microfluidic device, the first liquid, and the second liquid. FIG. 6 is a conceptual perspective view in an analysis process of a cell sorter or a flow cytometer which is an example of the present invention. FIG. 7 is (a) a conceptual perspective view and (b) an action diagram in a cell acquisition process of a cell sorter or a flow cytometer which is an example of the present invention. FIG. 8 is a schematic diagram showing the overall configuration of the cell sorter 1 in the cell sorter 1 as an example of the present invention. FIG. 9 is a functional block diagram of the cell sorter 1 which is an example of the present invention. FIG. 9 is a flowchart of the cell analysis control process in the cell sorter 1 which is an example of the present invention. FIG. 11 is a flowchart of the cell acquisition control process in the cell sorter 1 which is an example of the present invention. FIG. 12 is a flowchart according to a modified example of the cell analysis control process in the cell sorter 1 which is an example of the present invention. FIG. 13 is a diagram showing a synthesis scheme of the photodegradable hydrogel material used in the examples. FIG. 14 is a photograph showing that cells are stored and fixed in the microwell in Example 1. FIG. 15 is a fluorescence microscopic image of HL60 cells subjected to fluorescent staining immobilized on microwells in Example 2. FIG. 16 is a fluorescence microscopic image of fluorescent protein-expressing Ba / F3 cells immobilized in microwells in Example 3. FIG. 17 is a fluorescence microscopic image of fluorescent protein-expressing Ba / F3 cells immobilized in microwells in Example 4. FIG. 18 shows a conventional cell sorting technique.

Hereinafter, the present invention will be described more specifically. Here, the “subject” in the present invention is not particularly limited, and examples thereof include cells and beads (for example, beads carrying a target component such as a virus). However, in the following description, “cell” which is a preferred embodiment of the present invention will be described as an example of “subject”. Furthermore, the “degradable gel” in the present invention is not particularly limited as long as it is a gel that can be decomposed by applying a predetermined treatment, and is a gel that can be decomposed by energy (for example, light, sound, or heat), or a decomposition agent. The gel which can be decomposed | disassembled by addition can be mentioned. However, in the following description, “photodegradable gel” which is a preferred embodiment of the present invention will be described as an example of “degradable gel”. In addition, it demonstrates according to the following items. In addition, in the following description, even when described as “cell sorter” or “flow cytometer”, the term “flow cytometer” or “cell sorter” is also included.
(1. Cell sorting method)
1-1. Filling step 1-1-1. Microfluidic device structure 1-1-2. First liquid flowing in the microfluidic device 1-1-2-1. First component and second component, basic skeleton, part 1 / part 2, photodegradable part 1-1-3. Second liquid flowing into the microfluidic device 1-1-4. Process 1-2. Detection step 1-3. Analysis step 1-4. Acquisition process (2. Cell sorter)
2-1. Microfluidic device 2-2. Laser beam irradiation unit 2-3. Signal detection unit 2-4. Light irradiation part for gel decomposition

<< 1. Cell sorting method >>
The cell sorting method according to the present invention includes:
A flow step of flowing a liquid containing test cells into a flow path having a plurality of microwells, and filling the test cells in the microwells;
After the filling step, the signal detection step of irradiating the microwell with an information source wave (for example, laser light) and detecting a signal resulting from the irradiation;
Based on the signal in the signal detection step, an analysis step for analyzing the test cells filled in the microwells;
An acquisition step of taking out the test cells after the analysis step. Hereinafter, each process is explained in full detail.

<1-1. Filling process>
In the filling step, as described above, the first liquid containing the test cells is caused to flow through the flow path (microfluidic device) having the microwells, and the test cells are filled in the microwells. This is a step of removing the first liquid outside the microwell (the first liquid containing cells that have not entered the microwell or a liquid containing a part of the gel) by flowing the two liquids. The filling process will be described in detail with reference to FIGS.

{1-1-1. Microfluidic device structure}
An example of the microfluidic device A is shown in FIGS. Here, FIG. 1 is a cross-sectional view of the microfluidic device A, FIG. 2 is a top view of the microfluidic device A, and FIG. 3 is an exploded view of the microfluidic device. First, as can be understood from FIG. 1, the microfluidic device A has a planar structure in which a gap is provided between the upper surface A-1 and the lower surface A-2. As shown in FIGS. 1 and 3, the microfluidic device A has an insertion port Aa for introducing liquid into the gap (introduction for liquid insertion inserted into the insertion port Aa). Port A ₁ ) and an opening Ab for discharging the fluid in the gap (a discharge port A ₂ capable of sucking the liquid discharged from the opening Ab and discharging it to the outside) ing. 1 to 3, a large number of microwells A-3 are provided on the lower surface of the microfluidic device A. Here, in FIG. 3, the diameters of the microwells are 20 μm, 30 μm, and 40 μm, but are not limited thereto. The diameter of the microwell is appropriately selected depending on the size of the target cell, and is preferably 10 to 50 μm. In the case of general mammalian cells, the thickness is more preferably 15 to 30 μm. When the diameter is smaller than the preferred diameter, the cell filling rate is lowered, and when it is larger, a plurality of cells are filled into a single well, and the single cell filling rate is lowered. The well density in the microfluidic device A is not particularly limited, but is 100 to 10,000 / mm ² from the viewpoint of capturing single cells with high efficiency. In the case of general mammalian cells, the number is more preferably 500 to 2,000 cells / mm ² . If the density is lower than the preferred well density, the number of cells that can be processed in one analysis is reduced. Further, the upper limit of the well density is determined by the density when the preferred diameters are arranged in a close-packed manner. Further, the well interval is not particularly limited, but is preferably 2 μm or more. The microfluidic device includes at least a flow path through which a liquid can pass, a plurality of microwells provided on a wall surface of the flow path, an inlet for injecting fluid into the flow path, and the flow path An outlet for discharging fluid from the outlet. In addition, in order to prevent the adhesion of cells stored in the microwell, the microwell is surface-treated with a cell adhesion inhibiting component {for example, BSA, PEG, MPC (2-methacryloyloxyethyl phosphorylcholine), agarose, etc.}. It is preferable to do.

The height of the side wall (the distance between the upper surface A-1 and the lower surface A-2) can be appropriately designed according to the size of the well used, the properties of each liquid to be injected into the microfluidic device, the injection conditions, and the like. .

Further, as shown in FIGS. 1 and 3, the microfluidic device A has an upper surface A-1 and a lower surface A-2 formed of different members, and is interposed between the upper surface A-1 and the lower surface A-2. However, this is only an example, and the upper surface A-1, the lower surface A-2, and the side wall A-4 are integrally formed. There is no limitation as long as it can be used for the purpose. In this example, the upper surface A-1 and the lower surface A-2 of the microfluidic device A have a planar structure, but the present invention is not limited to this, and fine irregularities may be provided. As described above, since the lower surface A-2 of the microfluidic device A has a planar structure, the flow path of the first liquid containing the test cells is planar. As a result, the flow of the first liquid becomes smooth, and the microfluid can be more efficiently filled with the first liquid and the test cells. In this case, the microwell is preferably provided on the lower wall of the upper and lower walls (that is, the lower surface A-2 in this example) as in this example. By providing the microwell on the lower wall of the flow path, the filling of the test cell into the microwell can be ensured. Note that the microwell may be provided on the upper wall (upper surface A-1), the side wall A-4, or the like constituting the flow path according to the application.

{1-1-2. First liquid flowing in microfluidic device}
The first liquid that flows into the microfluidic device is a liquid that contains a plurality of test cells, and includes a first component and a second component that is different from the first component. Here, the first component and the second component react with time and gel. The first component and the second component exist in a state dissolved in a liquid (for example, a liquid in which cells can survive, for example, physiological saline or liquid medium). Hereinafter, each component contained in the first liquid will be described in detail.

(1-1-2-1. First component and second component)
As described above, the first component and the second component are gelled materials. Here, the first component has a first portion whose basic skeleton is a biocompatible polymer and can be bonded to the second component. The second component has a second part whose basic skeleton is a biocompatible polymer and can be bonded to the first component. Furthermore, the first component and / or the second component further has a photodegradable portion. Hereinafter, the “basic skeleton”, “first / second part”, and “photodegradable part” of the first and second components will be described in detail.

Basic skeleton The biocompatible polymer constituting the basic skeleton is not particularly limited, and is a carbohydrate-based polymer (methyl cellulose, carboxymethyl cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, ethyl cellulose, dextrin, cyclodextrin, alginate, hyaluronic acid and Chitosan, etc.); protein-based polymers (gelatin, collagen and glycol proteins, etc.); hydroxy acid polyesters (biodegradable polylactide-coglycolide (PLGA), polylactic acid (PLA), polyglycolide, polyhydroxybutyric acid, polycaprolactone, poly Valerolactone, polyphosphazene and polyorthoester, etc.); albumin; polyanhydride; polyethylene glycol Polyvinyl polyhydroxyalkyl methacrylates; pyrrolidone, polyvinyl alcohol. Of these, polyethylene glycol is preferred. In particular, multi-arm PEG (for example, 2-arm, 4-arm, 8-arm) is preferable. The weight average molecular weight of PEG is preferably 500 to 100,000, and more preferably 2,000 to 40,000. Since such PEG has little influence on the cells, it is excellent in that the cells can be collected in a form that does not impair the functions inherent to the cells (in other words, the cells can be collected while alive). Here, the weight average molecular weight is a value measured by MALDI-TOF-MS.

First part / second part The first part bonded to the basic skeleton of the first component (or the second part bonded to the basic skeleton of the second component) is the basic of the second component that constitutes the gel. It can be bonded to the second part bonded to the skeleton (or the first part bonded to the basic skeleton of the first component). Examples of such a combination of the first part and the second part include, for example, an azide group and an alkyne group (cycloaddition reaction), an azide group, which is a combination of reactive groups that form a chemical bond in liquid. Dibenzocyclooctyne group (cycloaddition reaction), thiol group and maleimide group (Michael addition reaction), thiol group and iodoacetamide group, thiol group and vinylsulfone group, aldehyde group and hydrazine group, ketone group and hydrazine group, aldehyde group And aminooxy groups, ketone groups and aminooxy groups, protein and ligand combinations that form chemical bonds or strong interactions, biotin and streptavidin, maltosyl and maltose binding proteins, glutathionyl and glutathione S-transferase, HaloTag® ligand and H loTag® protein, guanylylmethylphenyl group and SNAP-tag®, cytosynylmethylphenyl group and CLIP-tag, Strep-tag® and Strep-tactin®, antigen and Mention may be made of antibodies. In addition, visible light activated polymerization using a polymerizable monomer, acrylic group, methacrylic group, vinyl group, or epoxy group, as the first part and second part, and having a wavelength different from that of the photodegradable group It may be capable of binding by polymerization using any one of the initiators eosin Y, rose bengal, camphor-quinone, and erythrosine. Of the combinations described above, a combination of an azide group and an alkyne group is preferable because it hardly reacts with a functional group on the cell surface. The modification of the first part and the second part to the biocompatible polymer can be performed by a known method.

-Photodegradable part The photodegradable part may be present in one or both of the first component and the second component. Here, the photodegradable moiety refers to any group that can be eliminated by light irradiation. For example, nitrobenzyl group, nitrophenylethyl ester group (NPE), dimethoxynitrobenzyl ester group (DMNB), bromohydroxycoumarin (Bhc). ) Group, dimethoxybenzoin group, 2-nitropiperonyloxycarbonyl (NPOC) group, 2-nitroveratryloxycarbonyl (NVOC) group, α-methyl-2-nitropiperonyloxycarbonyl (MeNPOC) group, α -Methyl-2-nitroveratryloxycarbonyl (MeNVOC) group, 2,6-dinitrobenzyloxycarbonyl (DNBOC) group, α-methyl-2,6-dinitrobenzyloxycarbonyl (MeDNBOC) group, 1- (2- Nitrophenyl) ethyloxycarbonyl (NPEO) ) Group, 1-methyl-1- (2-nitrophenyl) ethyloxycarbonyl (MeNPEOC) group, 9-anthracenylmethyloxycarbonyl (ANMOC) group, 1-pyrenylmethyloxycarbonyl (PYMOC) group, 3 ′ -Methoxybenzoinyloxycarbonyl (MBOC) group, 3 ', 5'-dimethoxybenzoyloxycarbonyl (DMBOC) group, 7-nitroindolinyloxycarbonyl (NIOC) group, 5,7-dinitroindolinyloxycarbonyl (DNIOC) ) Group, 2-anthraquinonylmethyloxycarbonyl (AQMOC) group, α, α-dimethyl-3,5-dimethoxybenzyloxycarbonyl group, 5-bromo-7-nitroindolinyloxycarbonyl (BNIOC) group, etc. be able to. Among these, a group having a 2-nitrobenzyl derivative skeleton is suitable because it is not photodegraded by the degree of indoor lighting such as ordinary fluorescent lamps and incandescent lamps.

Here, FIG. 4 shows an example of a chemical structural formula of a bonding portion at the time of gelation between the first component and the second component, which are gelling components, and light irradiation (light having a wavelength at which the gel decomposes). ) Is an example of the chemical structural formula of the decomposition part. Here, the first component is 4-arm PEG-PL-azide (photodegradable 2-nitrobenzyl derivative skeleton is bonded to 4-arm PEG, and azide is further bonded to 2-nitrobenzyl derivative skeleton. The second component is 4-arm PEG-DBCO (dibenzylcyclooctyne linked to 4-arm PEG). After these are added and mixed in the liquid, a bridge is formed between the azide and DBCO.

In addition, the 1st liquid contains a 1st component and a 2nd component at the time of use as mentioned above. However, before use, that is, when the first liquid is transported or stored, the first component is preferably contained to prevent the gelation reaction between the first component and the second component before use. This is an embodiment of a kit comprising at least two liquids: a liquid and a liquid containing a second component.

{1-1-3. Second liquid flowing in microfluidic device}
After flowing the first liquid containing the test cells and the gelling material through the gap of the microfluidic device A, the first liquid is removed outside the microwell by flowing the second liquid. Although the 2nd liquid used in this case is not specifically limited, The component which is incompatible with 1st liquid, for example, oil, is suitable.

{1-1-4. process}
FIG. 5 is a diagram showing a state in which a test cell is fixed in a microwell by using the above device and solution. Referring to FIG. 5, the first liquid (gel solution) is injected into the inside from the introduction port of the microfluidic device ((a) in the figure). This operation is repeated a plurality of times (5 to 10 times in the example in the figure) ((b) in the figure). By this operation, cells can be filled into the microwell with high efficiency. However, the injection operation may be performed once. Thereafter, the second liquid (oil) is injected from the introduction port of the microfluidic device into the inside, and the excess first liquid existing inside is discharged out of the microfluidic device ((c) in the figure). Then, it incubates on predetermined conditions, the gel material (1st component and 2nd component) in a microwell is gelatinized, and the cell accommodated in the microwell is fix | immobilized.

In this embodiment, the first solution (gel solution) is filled in the microwells before gelation, but is not limited thereto. More specifically, the first liquid (gel solution) around the test cell is in a gelled state, and the first liquid (gel solution) is not gelated in the entire first liquid. It is preferable to inject from the introduction port of the microfluidic device. By setting it as such a process, the test cell can be filled in a microwell in the form which the gel around a test cell functions as a protective material, and does not impair the function which a cell originally has.

<1-2. Detection process>
Signal detection from a test cell fixed by a gel in a microwell can be the same as the detection method used in a conventionally known flow cytometry or cell sorter. For example, it is possible to acquire detection target information by irradiating a target wave with an information source and observing the response. As a more specific example, detection is obtained by irradiating a microwell to be detected with laser light (for example, single laser or dual laser such as argon, diode, die, helium neon, etc.), and resulting from the irradiation. Signal {forward scattered light (FSC), side scattered light (SSC), and when a target cell is previously labeled with a fluorescent substance, various fluorescence of the fluorescence-labeled cell} can be measured. It should be noted that such a wave serving as an information source is not particularly limited, and examples thereof include electromagnetic waves having appropriate wavelengths such as gamma rays to microwaves, and sound waves.

<1-3. Analysis process>
The analysis can be the same as the detection method used in a conventionally known flow cytometry or cell sorter. For example, the size of the cell, the complexity of the internal structure of the cell, and the like can be analyzed through analysis based on the signal (data) obtained in the detection step. In particular, according to the present invention, more detailed analysis such as cell imaging (for example, dynamic analysis of cell membrane molecules, analysis of cell chromosomes) can be performed. One feature of the present invention is that a test cell is fixed in a microwell, so that a plurality of items can be analyzed simultaneously.

<1-4. Acquisition process>
Acquisition is performed by irradiating light (wavelength light that decomposes the photodegradable gel in which the test cells are embedded in the microwells) to the microwells containing the test cells to be acquired. . The photodegradable gel in the microwell irradiated with the light is decomposed and the test cells in the microwell are fixed (falling below the microwell). Thereafter, by flowing a liquid (eg, physiological saline) through the flow path, the test cells are discharged together with the liquid (in addition, as the liquid released into the flow path in such an acquisition process, (Eg, physiological saline, liquid medium, etc.) can be suitably used. And the said to-be-detected cell discharged | emitted with the liquid is acquired. Thus, one feature of the present invention is that various types of desired cells can be selectively obtained from the same array.

In the above description, an example in which analysis is performed immediately after embedding cells in a microwell with a gel to obtain desired cells is not limited thereto. For example, after embedding the cells in a microwell with a gel, the cells may be cultured in the wells (for example, by culturing for a long time) by flowing a culture solution, and the cells after the culture may be analyzed. .

≪2. Cell sorter >>
Next, a cell sorter or a flow cytometer which is an example of the present invention will be described in detail with reference to FIGS. The basic configuration of the cell sorter or the flow cytometer is the same as that conventionally known. Therefore, the points different from the conventional one will be mainly described.

A cell sorter or flow cytometer 1 which is an example of the present invention,
A flow path (microfluidic device) 1-1 having a plurality of microwells 1-1-a;
A laser beam irradiation unit 1-2 capable of emitting a laser beam to one microwell of the plurality of microwells 1-1-a;
When the laser beam irradiation unit 1-2 irradiates the one microwell with a laser beam, a signal detection unit 1-3 for detecting a signal generated due to the irradiation;
Based on the signal from the signal detection unit 1-3, an analysis unit (not shown) for analyzing a test cell in the one microwell;
The laser beam irradiation unit 1-2, the signal detection unit 1-3, and the analysis unit (not shown) are configured to apply the irradiation and the processing to all of the plurality of microwells 1-1-a. A cell sorter 1 configured to perform detection and analysis,
The microwell 1-1-a can store a test cell and a photodegradable gel,
The cell sorter 1
After the analysis by the analysis unit (not shown), light for decomposing the photodegradable gel, which is irradiated with light having a wavelength different from that of the laser beam 1-4
It has further. Hereinafter, each part will be described in detail.

<2-1. Microfluidic device>
Since the microfluidic device has been described above, description thereof is omitted (although the microfluidic device A shown in FIGS. 1 to 3 and the microfluidic device 1-1 shown in FIGS. 6 and 7 have different shapes. , The function to have is the same). In addition, light irradiation (light irradiation from the laser beam irradiation unit 1-2 and / or the light irradiation unit for gel decomposition 1-4) and signal detection (signal irradiation) to the microwell 1-1-a in the microfluidic device 1-1 In order to perform the signal detection unit 1-3), it is desirable that at least a portion of the microfluidic device 1-1 where the microwell 1-1-a exists is light transmissive.

<2-2. Laser beam irradiation unit>
The laser beam irradiation unit 1-2 is configured to be able to irradiate each of the plurality of microwells 1-1-a independently. For example, in the example of FIG. 6, it is configured to be movable (front and rear, left and right) directly below (or directly above) each of the plurality of microwells 1-1-a (so-called scanning type). In this case, the signal detection unit 1-3, which will be described later, is also configured to be movable (front and rear, left and right) following the movement of the laser beam irradiation unit 1-2 (see FIGS. 6A and 6B). ). Note that the method of independently irradiating each of the plurality of microwells 1-1-a is not limited to this, and, for example, the laser beam irradiation unit 1-2 is irradiated by swinging under a fixed condition. A method of changing the positions of the microwells 1-1a to be performed, a method of providing the laser beam irradiation units 1-2 by the number of the plurality of microwells 1-1-a, and a plurality of microwells 1-1-a at a time. Irradiation method (for example, a method of forming a linear light source by arranging laser beam irradiation units in a line, or a surface that can irradiate light to a plurality of microwells in a certain area with a certain range as one section Or a method of irradiating a plurality of microwells all at once (using a light source capable of irradiating light to all the microwells). Further, when a plurality of analyzes are performed simultaneously, a plurality of laser beam irradiation units may be provided corresponding to the type of analysis. Further, the laser beam irradiation unit may be configured to change the type of detection wave and to perform a plurality of analyzes simultaneously. In this example, the light is irradiated from directly below (or directly above) the microwell 1-1-a. However, the light is irradiated obliquely below (or obliquely above) the microwell 1-1-a. The form which performs irradiation may be sufficient.

<2-3. Signal detection unit>
The signal detection unit 1-3 detects a signal emitted when the laser beam is irradiated to the test cell by the laser beam irradiation unit 1-2. Similar to the laser beam irradiation unit 1-2, it is necessary to detect the signals of the plurality of microwells 1-1-a. Therefore, in the example of FIG. 6, as described above, the movement of the laser beam irradiation unit 1-2 is performed. Is configured to be movable (front and rear, right and left) (see FIGS. 6A and 6B). Note that the method of detecting the signals of each of the plurality of microwells 1-1-a is not limited to this. For example, a method of providing the signal detection units 1-3 by the number of the plurality of microwells 1-1-a, A method for detecting signals from microwells (for example, a method in which a plurality of signal detectors 1-3 are arranged in a line, or a certain range as one section, and signals from a plurality of microwells in the one section are detected. Or a technique for detecting signals from a plurality of microwells at once. For example, when the signal detection unit is a microscope, the configuration can be changed as appropriate, such as imaging each microwell or imaging a plurality of microwells simultaneously. In addition, when performing a plurality of analyzes simultaneously, a plurality of signal detection units 1-3 may be provided corresponding to the type of analysis (for example, a plurality of signal detection units are provided in a form corresponding to one microwell. Installed). In this example, the signal detection unit 1-3 is shown on the opposite side of the laser beam irradiation unit 1-2 through the surface (flow path) where the microwell 1-1-a exists. However, the present invention is not limited to this, and can be appropriately changed depending on the type of signal to be detected, such as being on the same side as the laser beam irradiation unit 1-2.

<2-4. Light irradiation part for gel decomposition>
The gel-decomposing light irradiation unit 1-4 is configured to be able to irradiate each of the plurality of microwells 1-1-a independently. In FIG. 7, from the viewpoint of simplifying the drawing, it is described as the same as the laser beam irradiation unit 1-2, but the light of the laser beam irradiation unit 1-2 and the light beam irradiation unit for gel decomposition 1-4 Since the wavelength of this light is different, it is usually a different light source. However, in the case of a light source capable of changing the wavelength of light to be irradiated, it is not necessary to separate the laser beam irradiation unit 1-2 and the gel decomposition light irradiation unit 1-4. That is, in this case, the gel decomposition light irradiation unit and the laser beam irradiation unit may be the same light source. Further, a light source in which a plurality of light sources (laser beam irradiation unit 1-2 and gel decomposition light irradiation unit 1-4) are unitized may be used. Here, similarly to the laser beam irradiation unit 1-2, the gel-decomposing light irradiation unit 1-4 can also be moved directly below (or directly above) each of the plurality of microwells 1-1-a in the example of FIG. Front / rear / left / right). Note that, similarly to the laser beam irradiation unit 1-2 described above, the method of independently irradiating each of the plurality of microwells 1-1-a is not limited to this, and for example, the gel decomposition light irradiation unit 1-4 The method of changing the position of the microwell 1-1-a that is irradiated by swinging under a fixed state, or the gel-decomposing light irradiation unit 1-4 is connected to a plurality of microwells 1-1-a. A method of providing only the number may be used.

In addition, a confocal laser microscope etc. (CLSM) can be illustrated as a specific example of an apparatus which has a laser beam irradiation part and a signal detection part. More specifically, a plurality of cells are simultaneously imaged (microscopic analysis) with a confocal laser microscope to select the cells. Then, use the CLSM ROI (Region of Interest) mode to irradiate wells containing the target cells, and irradiate only that part with light of the photolysis wavelength (for example, 405 nm light) to decompose the gel, etc. do it.

In the cell sorter or flow cytometer of this example, various liquids can be sent from the inlet 1-1-b of the microfluidic device 1, and the decomposed gel or the like is discharged from the outlet 1-1 of the microfluidic device 1. -C is configured to be discharged from the outlet, and a box 1-6 is provided at the outlet 1-1-c for collecting the discharged liquid and the cells contained in the gel (such liquid feeding, Ejection, cell recovery, etc. may be performed by appropriate means as necessary).

<2-6. Analysis flow>
Next, the flow of analysis by the cell sorter or flow cytometer of the present invention (hereinafter simply referred to as cell sorter 1 etc.) will be described. Here, analysis is performed when the detection step and the analysis step are performed in a state where the microfluidic device 1-1 having a microwell filled with a gel containing a test cell is installed at a predetermined position of the cell sorter 1. This flow will be described as an example.

The cell sorter 1 of this example includes a laser beam irradiation unit 1-2 (laser irradiation unit 1-2) and a laser irradiation unit capable of laser beam irradiation corresponding to one microphone well shown in FIGS. A signal detection unit 1-3 capable of detecting a signal derived from the light irradiation of 1-2 and a light irradiation unit for gel decomposition 1-4 capable of light irradiation corresponding to one microphone well, It is configured to be movable (front / back / left / right).

First, FIG. 8 is a schematic diagram showing the overall configuration of the cell sorter 1 of the present invention. As shown in the figure, the cell sorter 1 includes a processing unit 100 having a CPU, a ROM area, and a RAM area, a signal detection unit 1-3, a laser irradiation unit 1-2, and a gel decomposition light irradiation unit 1. -4, a phase liquid unit 2, a cell recovery unit 3, and a display unit 4.

Here, the liquid feeding unit 2 is a device capable of filling various liquids (the liquids such as the first liquid, the second liquid, and the physiological saline described above) in the microfluidic device 1-1, and the inside of the apparatus. By changing the flow path, the liquid can be appropriately selected and switched. The cell collection unit 3 is an apparatus (for example, the above-described box 1-6) that can collect cells discharged from the microwell 1-1-a. The display unit 4 is a device that can display various data (for example, cell information described later) of the processing unit 100 as an image. Other configurations are the same as described above, and will be omitted.

Next, FIG. 9 is a functional block diagram of the cell sorter 1 of this example. As shown in the figure, the processing unit 100 includes a detection device control unit 110 capable of controlling the device related to the analysis process / detection step, a gel decomposition device control unit 120 capable of controlling the device related to the acquisition process, and a transmission unit. The liquid feeding control unit 130 that can control the liquid unit 2 and the cell collection control unit 140 that can control the cell acquisition behavior of the cell collection unit 3 (for example, opening / closing of the opening for introducing cells, the position of the opening, etc.). The information storage unit 150, the cell information determination unit 160 capable of making various determinations based on the cell information stored in the information storage unit 150, and the display control capable of controlling the video output of the display unit 4 Part 170. As shown in this figure, in this example, the detection device having the laser irradiation unit 1-2 and the signal detection unit 1-3, the gel decomposition device having the gel irradiation light irradiation unit 1-4, The liquid unit 2, the cell collection unit 3, and the display device 4 are electrically connected to the processing unit 100.

The detection device control unit 110 also controls the laser irradiation control unit 111-1 that can control the laser irradiation mode (irradiation time, irradiation intensity, etc.) in the laser irradiation unit 1-2, and the microwell of the laser irradiation unit 1-2. A laser irradiation position control unit 111-2 capable of controlling the irradiation position, a signal detection control unit 121-1 capable of controlling a detection mode (detection time, etc.) of the signal detection unit 1-3, and a signal detection unit 1-3 A signal detection position control unit 112-2 capable of controlling the detection position with respect to the microwell.

The gel decomposition apparatus control unit 120 includes a gel decomposition light irradiation control unit 121-1 capable of controlling the light irradiation mode (irradiation time, irradiation intensity, etc.) in the gel decomposition light irradiation unit 1-4, and a gel decomposition light source. A gel-decomposing light irradiation control unit 121-2 capable of controlling the irradiation position of the light irradiation unit 1-4.

Next, FIG. 10 is a flowchart relating to cell analysis control processing (step 1000) in the cell sorter 1.

First, in a state where the microfluidic device 1-1 is installed at a predetermined position of the cell sorter 1, in step 1001, the processing unit 100 determines whether cell analysis control is started. In the case of Yes in step 1001, in step 1002, the liquid feeding control unit 130 feeds the cell-containing liquid (first liquid) from the liquid feeding unit 2 into the microfluidic device 1-1, and the microfluidic device 1- Fill the microwell in 1 with the cell-containing solution. Next, in step 1004, the liquid feeding control unit 130 feeds oil from the liquid feeding unit 2 into the microfluidic device 1-1, and eliminates excess cell-containing liquid in the microfluidic device 1-1. (Note that the liquid fed in Step 1004 is not limited to oil, and may be the second liquid described above). Next, in step 1006, the processing unit 100 performs initial setting processing of the laser irradiation unit 1-2 and the signal detection unit 1-3 (in this example, the variable n and each microwell in the microfluidic device 1-1). 1 is substituted for n in a situation where the position information is associated with the position information). Next, in step 1008, the laser irradiation position control unit 111-2 and the signal detection position control unit 112-2 move the laser irradiation unit 1-2 and the signal detection unit 1-3 to the position n. Next, in step 1010, the laser irradiation control unit 111-1 irradiates the microwell existing at the position n from the laser irradiation unit 1-2 (at this time, the signal detection unit 1- 1). 3 detects the signal obtained when the detection laser is irradiated). Next, in step 1012, the processing unit 100 temporarily stores cell information related to cells existing in the microwell irradiated with the detection laser in the information storage unit 150 based on the signal detected by the signal detection unit 1-3. (In this case, as an example, even if there is no cell in the well, the cell information is temporarily stored. However, if there is no cell in the well, cell information on the corresponding microwell is stored. May not be stored). Next, in step 1014, the processing unit 100 determines whether or not irradiation of the detection laser to all the microwells has been completed (whether n has reached fin). If Yes in step 1014, the process proceeds to step 1018. On the other hand, in the case of No in step 1014, in step 1016, the processing unit 100 adds 1 to n and proceeds to step 1008. Next, in step 1018, the cell information determination unit 160 refers to all the temporarily stored cell information {in this example, the number of cell information equal to the total number (n) of microwells} and is set in advance. Based on the determination information, it is determined whether each cell is a desired cell. Next, in step 1020, the cell information determination unit 160 determines whether or not a desired cell exists in the microwell. In the case of Yes at step 1020, the processing unit 100 temporarily stores the position information of the microwell where the desired cell exists in the information storage unit 150, and proceeds to step 2000.

On the other hand, in the case of No in step 1001 and step 1020, the cell analysis control process (step 1000) is terminated.

As described above, when performing analysis using a plurality of types of detection lasers by providing a plurality of laser irradiation units 1-2 or changing the laser irradiation conditions, step 1006 to step 2000 are performed. You may repeat until.

Here, in this example, the variable n and the position information of each microwell in the microfluidic device 1-1 are associated, but the position information of the microwell may be the position information of the microwell itself. However, the surface on which the microwell is provided may be appropriately classified (grouped), and information relating to the classification may be used, and is not limited at all. Further, such microwell position information may be incorporated in advance in the processing unit 100, or the microwell position may be automatically determined by an optical method or the like.

When moving the laser irradiation unit 1-2, the configuration of the present example in which the laser irradiation unit 1-2 moves to the next microwell after completing the analysis for one microwell (the laser irradiation unit 1-2). It is not limited to the configuration in which the stop and the movement are repeated. For example, the laser irradiation unit 1-2 may be configured to be able to move continuously across a plurality of microwells (a configuration in which a certain range of analysis is performed at a constant speed like a scanner). In this case, since the position where the desired cell information is acquired can be read as the microwell position information even if the microwell position does not exist, the setting of the microwell position information in advance is not necessary ( The analysis start condition and end condition may be changed as appropriate).

In this example, it is assumed that one laser irradiation is performed once for one microwell. However, as described above, the configuration allows analysis of a plurality of microwells by one laser irradiation. May be. In particular, when there are a plurality of laser irradiation portions corresponding to each microwell one-to-one, or when the configuration allows analysis of all the plurality of microwells by one laser irradiation, the laser irradiation portion 1-2 is used. Since all the cell information can be acquired with the laser beam stopped, the laser irradiation unit 1-2 does not need to move. In this example, the laser irradiation unit 1-2 itself moves. However, as described above, the laser irradiation unit 1-2 is swung, or the irradiation angle is changed by a reflecting plate. A configuration may be adopted in which the microwell for analysis is changed.

Further, as described above, the detection laser emitted from the laser irradiation unit 1-2 used in the analysis step is not limited to laser light, and has a function capable of irradiating an appropriate information source wave capable of acquiring cell information. There is no limitation at all. Further, as described above, the laser irradiation unit 1-2 has a configuration capable of oscillating a plurality of information source waves (that is, a configuration capable of changing the oscillation wavelength and frequency, and a configuration including a plurality of oscillation terminals). Alternatively, a plurality of laser irradiation units 1-2 may be provided depending on the type of information source wave to be oscillated.

In addition, when the configuration of the laser irradiation unit 1-2 is changed, the configuration of the signal detection unit 1-3 may be changed as appropriate, and there is no limitation as long as signal detection is possible. For example, the cell information related to all the plurality of microwells may be configured to be detected by a single fixed signal detection unit 1-3 (in this case, the signal detection unit 1-3). The control related to the movement is unnecessary.) In the case of performing analysis using a plurality of information source waves, the signal detector 1-3 may be configured to have one signal detector 1-3 for one information source wave, or one signal The detection unit 1-3 may be configured to detect a plurality of signals.

Here, in the cell analysis control process (step 1000), after step 1004, in order to gel the components in the cell-containing liquid (first liquid), a subsequent process may be waited (or Or a treatment for promoting gelation may be interposed). In addition, after preparing a plurality of microfluidic devices 1-1 and executing the processing according to step 1002 and step 1004 for one microfluidic device 1-1, successively for another microfluidic device 1-1. The processing according to step 1002 and step 1004 may be executed. In other words, the processing according to Step 1002 and Step 1004 can be executed for another microfluidic device 1-1 during the waiting time required for gelation of components in the cell-containing liquid in one microfluidic device 1-1. It is good. In this case, the components in the cell-containing liquid in the microfluidic device 1-1 may be shifted to the next process sequentially from the gelled component.

Next, FIG. 11 is a flowchart of the cell acquisition control process (step 2000) in the cell sorter 1.

First, in step 2002, the processing unit 100 refers to the information storage unit 150, and reads position information of a predetermined microwell that is determined to have a desired cell. Next, in step 2004, the processing unit 100 performs an initial setting process for the gel-decomposing light irradiation unit 1-4 (the variable m is linked to the position information of a predetermined microwell in the microfluidic device 1-1). Under the circumstances, 1 is substituted into m). In this example, among the predetermined well position information read out in step 2002, the most upstream position of the microfluidic device 1-1 is set as the initial position (m = 1). Next, in step 2006, the gel-decomposing light irradiation position control unit 121-2 moves the gel-decomposing light irradiation unit 1-4 to the position m. Next, in step 2008, the gel decomposition light irradiation control unit 121-1 irradiates the gel decomposition laser to the microwell existing at the position m from the gel decomposition light irradiation unit 1-4. Next, in step 2010, the liquid feeding control unit 130 sends a liquid (eg, physiological saline) from the liquid feeding unit 2 into the microfluidic device 1-1 as a liquid feeding process (at this time, it is decomposed). And the cells contained in the gel are transferred out of the microwell). Next, in step 2012, the cell recovery control unit 140 controls the cell recovery unit 3 as a cell acquisition process, and together with the fed liquid, the decomposed gel and the cells contained in the gel are stored in the cell recovery unit. Collect in 3. Next, in step 2014, the processing unit 100 determines whether or not the gel decomposition laser has been irradiated to all the predetermined microwells. If Yes in step 2014, the cell acquisition control process 2000 ends. On the other hand, in the case of No in step 2014, in step 2016, the processing unit 100 adds 1 to m, and proceeds to step 2006.

In this example, it is assumed that one microwell is irradiated with a single laser for gel decomposition. However, when there are a plurality of laser irradiation sections corresponding to each microwell, a single laser is used. The structure which can perform the gel decomposition | disassembly with respect to several microwells by irradiation may be sufficient. In addition, in this example, the gel decomposition light irradiation unit 1-4 itself moves, but as described above, the gel decomposition light irradiation unit 1-4 is swung or irradiated by a reflector. The microwell for performing gel decomposition may be changed by changing the angle or the like (in this case, gel decomposition can be performed in all wells while the gel decomposition light irradiation unit 1-4 is stopped). The gel-decomposing light irradiation unit 1-4 may not move).

In this example, the cell analysis control process (step 1000) and the cell acquisition control process (step 2000) are continuous processes, but these may be independent processes.

In addition, in this example, although an example of the system which performs a detection process and an analysis process collectively and automatically was demonstrated, it is not limited to this, In part, it is a system with manual processing. Also good. An example of such a system will be described below.

Next, FIG. 12 is a flowchart of the cell analysis control process (S1000-2) according to a modified example of the cell analysis control process (S1000) in the cell sorter 1 which is an example of the present invention. Here, only the changes from the cell analysis control process (S1000) will be described.

First, if Yes in step 1014, the process proceeds to step 1019-2. Next, in step 1019-2, the display control unit 170 refers to the information storage unit 150 and displays information related to cell information on the display device 4. Next, in step 1020-2, the processing unit 100 determines the desired cell in the microwell based on selection information described later (in this example, information input to the processing unit 100 by the operator using an input terminal or the like). It is determined whether or not it is determined that exists. If Yes in step 1020-2, the process proceeds to cell acquisition control processing (step 2000).

In this modification, the process can be interrupted between step 1019-2 and step 1020-2. Before step 1020-2, that is, while the processing is interrupted, the operator observes the cell information displayed on the display device 4 (for example, display), for example, and whether or not a desired cell exists. Judgment is made. The selection information (whether there is a predetermined microwell in which a desired cell exists and the position of the predetermined microwell, etc.) related to the result of the determination is processed by the processing unit 100 (information storage unit 150) using an input terminal or the like. And is referred to by the processing unit 100 in step 1020-2. As described above, when the cell analysis control process (step 1000-2) is performed (the operator performs determination regarding cell information), the processing unit 100 may not include the cell information determination unit 160.

As described above, the cell sorter 1 may be in a mode in which a manual operation is performed on a part of the cell sorter 1 (a mode in which the operator appropriately determines and performs various processes based on the determination). For example, a cell-containing liquid feeding process, an oil feeding process, a liquid feeding (for example, physiological saline) process, cell acquisition, and the like may be performed manually as appropriate.

Hereinafter, the present invention will be described more specifically with reference to examples. In addition, this invention is not limited to an Example.

<Example 1>
(Synthesis of gel material)
In general, the gel material used in this example was synthesized based on the synthesis scheme of FIG. Hereinafter, a detailed synthesis method of 4-arm PEG-azide and 4-arm PEG-DBCO, which are gel materials used in this example, will be described. The number of repetitions n of the starting 4-arm PEG-amine is about 111.

・ Synthesis of Compound 2

The reaction was performed in the dark. In a 50 mL branch flask, 1.15 g (10.0 mmol, 2 eq) of N-Hydroxysuccinimide (NHS) and 1.99 g (10.0 mmol, 2 eq) of 1-Ethyl-3- (3-dimethylaminopropyl) carbodiimide Hydrochloride (EDC) The mixture was dried up for 20 minutes, purged with nitrogen, and 18 mL of dry DMF was added. After stirring at room temperature to dissolve NHS and EDC, 1.51 g (5.01 mmol, 1 eq) of 4- [4- (1-Hydroxyethyl) -2-methoxy-5-nitrophenoxy] butyric acid (1) was added, Further, 2 mL of dry DMF was added. After stirring at room temperature for 10.5 hours, the completion of the reaction was confirmed, and the solution was concentrated with an evaporator. The concentrated solution was dark yellowish brown, pure water was added, and the precipitated pale yellow solid was collected by suction filtration. A pale yellow solid was obtained after vacuum drying overnight. The yield was 1.97 g, and the yield was 103%. The reason why the yield exceeded 100% is considered that DMF as a solvent could not be completely removed. The product was identified by ¹ H-NMR (CDCl ₃ ). ¹ H NMR (600 MHz, CDCl ₃ , TMS) δ = 7.59 (s, 1H), 7.31 (s, 1H), 5.57 (q, J = 5.9 Hz, 1H), 4.18 (t, J = 6.2 Hz, 2H ), 3.99 (s, 3H), 2.90 (t, J = 8.5 Hz, 2H), 2.86 (br s, 4H), 2.29 (quin, J = 6.2 Hz, 2H), 1.56 (d, J = 6.4 Hz, 3H)
Synthesis of compound 3

The reaction was conducted in the dark and under an argon atmosphere. In a 50 mL flask, 1.11 g (2.80 mmol, 1 eq) of Compound 2 was added and dried up, and then placed in an argon atmosphere. 10 mL of dry DMF was added, and the mixture was stirred at room temperature. The solution was orange transparent. When 0.700 g (7.00 mmol, 2.5 eq) of 3-azidopropyl-1 -amine was added and reacted at room temperature for 18 hours, the solution was subjected to an evaporator to obtain an orange transparent oil. This was vacuum dried overnight. The mixture was diluted with 100 mL of ethyl acetate and washed three times with pure water. The organic phase was dried over MgSO ₄ and filtered, after which the solution was evaporated to give an orange clear oil. Vacuum drying was performed to obtain a brown solid. The yield was 0.97 g and the yield was 91%. The product was identified by ¹ H-NMR (CDCl ₃ ). ¹ H NMR (600 MHz, CDCl ₃ , TMS) δ = 7.56 (s, 1H), 7.31 (s, 1H), 5.91 (brs, 1H), 5.56 (q, J = 10.3 Hz, 1H), 4.11 (t , J = 8.8 Hz, 2H), 3.99 (s, 3H), 3.35 (dt, J = 10.3 Hz, 4H), 2.42 (t, J = 10.3 Hz, 2H), 2.20 (quin, J = 8.8 Hz, 2H ), 1.78 (quin, J = 10.3 Hz, 2H), 1.56 (d, J = 10.2 Hz, 3H)
Synthesis of compound 4

The reaction was conducted in the dark and under an argon atmosphere. In a 10 mL two-necked flask, 61.7 mg (0.17 mmol, 1 eq) of PL-azide (3) and 112 mg (0.79 mmol, 3.3 eq) of 4-Nitrophenyl Chloroformate were added, dried, and placed in an argon atmosphere. When 2 mL of dry CH ₂ Cl ₂ was added and stirred, an orange transparent solution was obtained. To this, 110 mL (d = 0.726 g / cm ³ , 0.80 mmol, 4.7 eq) of dry triethylamine was added and stirred for 1.5 hours. As a result, disappearance of compound 3 as a raw material was confirmed. When directly connected to a vacuum pump and triethylamine and CH ₂ Cl ₂ were distilled off, a yellowish green oil was obtained. Silica gel column chromatography was performed using hexane: ethyl acetate = 1: 1 → ethyl acetate 100%. The column diameter was 2 cm and the column length was 10 cm. The fraction containing the desired product was applied to an evaporator and vacuum-dried to obtain a pale yellow solid. The yield was 46.3 mg, and the yield was 50%. The product was identified by ¹ H-NMR (CDCl ₃ ) and ESI-MS (pos). ¹ H NMR (600 MHz, CDCl ₃ , TMS) δ = 8.26 (d, J = 9.1 Hz, 2H), 7.62 (s, 1H), 7.35 (d, J = 9.1 Hz, 2H), 7.12 (s, 1H ), 6.53 (quin, J = 6.4 Hz, 1H), 5.82 (brs, 1H), 4.14 (t, J = 6.2 Hz, 2H), 4.01 (s, 3H), 3.36 (m, 4H), 2.42 (t , J = 7.1 Hz, 2H), 2.21 (quin, J = 6.5 Hz, 2H), 1.79 (m, 5H)
・ Synthesis of 4-arm PEG-azide

The reaction was conducted in the dark and under an argon atmosphere. In a 10 mL two-necked flask, add 152 mg (7.5 mmol, 1 eq) of PTE-200PA (SUNBRIGHT, terminal NH ₂ 4-branched PEG, Mw to 20000) and 46.3 mg (84.7 mmol, 11 eq) of compound 4 and dry. Up and under an argon atmosphere. After adding 1.5 mL of dry CH ₂ Cl ₂ and stirring at room temperature for 10.5 hours, disappearance of PTE-200PA as a raw material was confirmed. When the solution was added dropwise to a tube containing 30 mL of diethyl ether on an ice bath, a pale yellow precipitate was formed. The tube was centrifuged (4 ° C, 10 krpm, 10 min), the supernatant was decanted and allowed to stand at room temperature, and then vacuum dried in a desiccator to remove diethyl ether. Add 3 mL of 50 mM Tris-HCl buffer (pH 8.0) and 3 mL of MilliQ, centrifuge the tube (4 ° C, 5000 rpm, 3 min), and add the supernatant to MiiliQ 1.8 L, molecular weight fraction 3500. The dialysis membrane was dialyzed for 3.5 days. The white solid of the desired 4-arm PEG-PL-azide was obtained by lyophilization. The yield was 80 mg and the yield was 49%. Identification and molecular weight measurement of the product were performed by ¹ H-NMR (CDCl ₃ ) and MALDI-TOF MS (matrix: sinapinic acid), respectively. ¹ H NMR (600 MHz, CDCl ₃ , TMS) δ = 7.58 (s, 4H), 7.02 (s, 4H), 6.34 (m, 4H), 6.00 (brs, 4H), 5.52 (brs, 4H), 3.25 -4.11 (brm), 2.41 (m, 8H), 2.20 (m, 8H), 1.60-1.88 (brm, 12H + H ₂ O)
・ Synthesis of 4-arm PEG-DBCO

The reaction was performed under a nitrogen atmosphere. Add 15 mg (7.5 mmol, 1 eq) of PTE-200PA and 21.5 mg (52.5 mmol, 7 eq) of DBCO NHS-ester to a 15 mL two-necked flask, dry it up, replace with N ₂ , and then dry CH ₂ Dissolved in 2 mL of Cl _{2 and} stirred at room temperature for 18 hours. When the disappearance of PTE-200PA as a raw material was confirmed, the reaction solution was put into two 50 mL centrifuge tubes each containing 20 mL of diethyl ether in an ice bath and subjected to ether precipitation. As a result, a white precipitate was formed. After centrifuging at 4 ° C and 10 krpm for 7 minutes, the supernatant was removed by decantation, left to dry at room temperature, and further dried in a desiccator under vacuum. To each tube, 3 mL of 50 mM Tris / HCl (pH = 8.0) buffer prepared and 3 mL of MilliQ were added and suspended. The solution was centrifuged at 3 ° C. for 3 minutes at 4 ° C., and dialyzed against MilliQ 2 L using a dialysis membrane of 3500 molecular weight fraction. Water exchange was performed after 19.5 hours and after 22.5 hours. Lyophilization was performed to obtain a white solid. The yield was 130 mg, and the yield was 83%. The product identification and molecular weight measurement were performed by ¹ H-NMR (CDCl ₃ ) and MALDI-TOF-MS (matrix: sinapinic acid), respectively. ¹ H NMR (600 MHz, CDCl ₃ , TMS) δ = 7.68 (d, J = 7.3 Hz, 4H), 7.52 (d, J = 8.2 Hz, 4H), 6.23 (brs, 4H), 5.15 (d, J = 14.1 Hz, 4H), 3.25-4.65 (brm), 3.22 (q, J = 5.9 Hz, 8H), 2.79 (m, 4H), 2.42 (m, 4H), 2.17 (m, 4H), 1.95 (m , 4H)
(Preparation of cell-containing solution)
After placing a plurality of calcein-stained Ba / F3 cells in physiological saline, the two kinds of gel materials (4-arm PEG-PL-azide and 4-arm PEG-DBCO) shown in FIG. A liquid was obtained. The cell density is 50,000,000 cells / mL, and the concentration of the gel material is 0.9 w / v%.
(Cell fixation in microwells)
The prepared cell-containing liquid was injected from the inlet port A ₁ of the microfluidic device A to the device (microwell diameter = 20 [mu] m) in shown in FIG. The operation was performed a plurality of times. Then, it incubated at 4 degreeC for 5 minute (s), and the cell was settled in the microwell. Then poured Fluorinert FC-40 (trade name) from the inlet A ₁ in the device, was discharged those present in excess to the outside of the micro-well from the outlet A _2. Then, it incubated at 4 degreeC for 20 minute (s), and the cell containing liquid in a microwell was gelatinized. As a result, as shown in FIG. 14, it was confirmed that cells were accommodated in 64% of the microwells.
(Analysis of cells fixed in microwells)
Fluorescence emitted by irradiating a calcein-stained Ba / F3 cell fixed to a microwell with a laser was measured, and the size and shape of the cell were analyzed.
(Removal of cells fixed in microwells)
After the cell analysis, light having a wavelength of 405 nm was irradiated to a specific microwell on which the cell was fixed, and the gel in which the cell was embedded was decomposed. Thereafter, flushed with saline from the inlet A ₁ of the microfluidic device A. As a result, the cells fixed in the specific microwell could be selectively obtained.
<Example 2>
HL60 cells subjected to calcein staining were immobilized in the same manner as in Example 1, except that a photodegradable hydrogel modified with a red fluorescent dye was used. Thereafter, light (405 nm) was irradiated to specific cell groups, and the gels in these microwells were dissolved. Thereafter, these cells were selectively obtained by flowing a liquid (buffer solution under physiological conditions). Here, FIG. 15a is an image of a cell immobilized in a microwell with a photodegradable hydrogel (superimposed image of a green fluorescence and a red fluorescence image and a bright field image), and a portion surrounded by a square is light. It is a microwell irradiated with (405 nm). FIG. 15b is an image of cells immobilized with a photodegradable hydrogel in a microwell after being washed a predetermined number of times (in this example, 15 times) with a liquid (buffer solution under physiological conditions) after light irradiation. is there. As can be seen from FIG. 15b, in this example, a plurality (seven cells in this example) of cell groups could be obtained collectively.
<Example 3>
Ba / F3 cells expressing green fluorescent and red fluorescent proteins (EGFP and Kusabila-Orange) were tested in the same manner as in Examples 1 and 2. FIG. 16 is a fluorescence microscopic image of fluorescent protein-expressing Ba / F3 cells immobilized in a microwell {a is a superimposed image (low magnification) of a green fluorescent image and a red fluorescent image, b is Superposed image (high magnification) of green fluorescence and red fluorescence image and bright field image}. Although it is difficult to understand from the figure, there are microwells that emit only green fluorescence, microwells that emit only red fluorescence, and microwells that emit red and green fluorescence. The reason why red fluorescence is emitted from cells mainly emitting green is that light emitted as scattered light from the cells themselves was detected when laser light emitting red fluorescence was irradiated. On the contrary, when the laser beam emitting green fluorescence is irradiated, green fluorescence is emitted only from cells expressing EGFP, and no green fluorescence is emitted from cells expressing Kusabila-Orange. In addition, although there are a few microwells containing two cells, such microwells can be identified by comparing a fluorescent image and a bright field image.
<Example 4>
With respect to the fluorescent protein-expressing Ba / F3 cells prepared in Example 3 and immobilized in a photodegradable hydrogel in a microwell, only the cells having red fluorescence were selectively removed (first stage), and then (2nd stage) Only cells having green fluorescence were selectively removed. Here, FIGS. 17a to 17d are images of cells immobilized with a photodegradable hydrogel in a microwell (superposed images of green fluorescence and red fluorescence images and bright field images). Although it is difficult to understand from the figure, there are microwells that emit only green fluorescence, microwells that emit only red fluorescence, and microwells that emit red and green fluorescence. FIG. 17a is an image before selective extraction of the first-stage red fluorescent cells (a portion surrounded by a square = a well in which red fluorescent cells are present), and FIG. 17b is a first-stage red fluorescent cell. This is an image after selective extraction (portion surrounded by a square = well in which red fluorescent cells were present). Further, FIG. 17c is an image before selective extraction of the second stage green fluorescent cells (portion surrounded by a square = well in which green fluorescent cells are present), and FIG. 17d is the second stage green fluorescent cells. This is an image after selective extraction (portion surrounded by a square = well in which green fluorescent cells were present).

Claims

A flow path having a plurality of microwells;
An introduction path capable of introducing a liquid containing a plurality of analytes into the flow path;
An information acquisition unit for acquiring information from a plurality of subjects stored in the plurality of microwells;
Based on the information acquired by the information acquisition unit, a selective extraction means capable of selectively extracting a subject in one microwell from the microwell;
A cell sorter or a flow cytometer having a specimen collecting section capable of collecting a specimen selectively taken out by the selective taking-out means,
The plurality of microwells can store the analyte and the degradable gel,
A cell sorter or flow cytometer configured such that the selective extraction means is a decomposition processing unit that performs a process of decomposing the degradable gel in the microwell in which the specimen to be extracted is held.
The cell sorter or flow cytometer according to claim 1, wherein the flow path is planar, and the plurality of microwells are provided on a lower wall constituting the flow path.
The degradable gel is a photodegradable gel;
The cell sorter or flow cytometer according to claim 1 or 2, wherein the decomposition processing unit is a light irradiation unit capable of irradiating light having a wavelength at which the photodegradable gel is decomposed.
The information acquisition unit
The information from the plurality of microwells can be acquired for each microwell, acquired at one time for two or more of the plurality of microwells, or acquired at once for all the plurality of microwells. The cell sorter or flow cytometer according to any one of claims 1 to 3.
The information acquisition unit
An information source wave irradiation unit capable of irradiating the plurality of microwells with an information source wave serving as a source of the information to be acquired;
5. An information receiving unit that receives the information that can be generated from the plurality of microwells when the information source wave irradiation unit irradiates the information source waves to the plurality of microwells. The cell sorter or flow cytometer according to any one of the above.
The information source wave irradiation unit can irradiate the information source wave for each microwell as the first aspect; as the second aspect, it can irradiate two or more of the plurality of microwells at a time. Or, as a third embodiment, all of the plurality of microwells can be irradiated at once; in the case of the first embodiment and the second embodiment, all of the plurality of microwells are scanned and irradiated. The cell sorter or flow cytometer according to claim 5 obtained.
The cell sorter or flow cytometer according to claim 5 or 6, wherein the information source wave irradiation unit is capable of outputting a plurality of types of information source waves according to the number of analysis items.
The information source wave irradiated from the information source wave irradiation unit is light, the degradable gel is a photodegradable gel, and the decomposition processing unit is light having a wavelength at which the photodegradable gel is decomposed. The cell sorter or flow cytometer according to any one of claims 5 to 7, wherein when the light irradiation unit is capable of irradiating, the wavelength of the light is different from the wavelength of the light decomposed by the photodegradable gel.
The light irradiation unit and the information source wave irradiation unit are the same light source,
The same light source can switch and irradiate light having a wavelength at which the photodegradable gel decomposes and light having a wavelength different from the wavelength at which the photolytic gel is decomposed, which is a source of the information to be acquired. The cell sorter or flow cytometer according to claim 8, which is configured as follows.
The cell sorter or flow cytometer is
It may have an automatic analysis means for automatically analyzing the information acquired by the information acquisition unit,
Furthermore, based on the user's instruction or the analysis result by the automatic analysis means, the selective extraction means is controlled so that the degradable gel in the microwell containing the specimen to be obtained is subjected to a process for degrading the gel. The cell sorter or flow cytometer according to any one of claims 1 to 9, further comprising a selective take-out control means.
A microfluidic device comprising the flow path, which is for the cell sorter or flow cytometer according to any one of claims 1 to 10.
A liquid containing a material capable of forming the degradable gel, which is for the cell sorter or flow cytometer according to any one of claims 1 to 10.
A filling step of flowing a liquid containing a specimen into a flow path having a plurality of microwells, and filling the specimen into the microwell;
After the filling step, an analysis step for analyzing the specimen filled in the microwell;
After the analysis step, an object recovery method including an acquisition step of taking out the subject,
The liquid contains a first component and a second component different from the first component,
The first component has a first portion in which the basic skeleton is a biocompatible polymer and can be bonded to the second component;
The second component has a second part in which the basic skeleton is a biocompatible polymer and can bind to the first component;
In the analysis step, a gel in which the first component and the second component are cross-linked is formed in the microwell by the bond formation between the first part and the second part. ,
The first component and / or the second component further has a degradable portion,
In the acquisition step, a subject recovery method in which, by performing a decomposition process, the degradable portion is decomposed to decompose the gel, and the sample is taken out.
14. The specimen recovery method according to claim 13, wherein the first part is an azide and the second part is an alkyne.