JP2019090610A - Cell sorting method, and flow cytometry and cell sorter using the same - Google Patents

Abstract

To provide a cell acquisition technique which can analyze a plurality of cells simultaneously and can be automated while reducing the frequency of false positives and false negatives.SOLUTION: A cell sorter or a flow cytometer is comprised of: a channel 1-1 having a plurality of microwells 1-1-a; an introduction path which can introduce a solution containing a plurality of test cells into the channel; an information acquisition unit which acquires information from the plurality of test cells stored in the plurality of microwells; selective extraction means which can selectively extract test cells in one microwell from the microwells on the basis of the information acquired by the information acquisition unit; and a test cell recovery unit which can recover the test cells selectively extracted by the selective extraction means. The selective extraction means is a decomposition light irradiation unit which irradiates light having a wavelength in which photodegradable gel is decomposed with respect to the photodegradable gel in the microwell in which the test cells to be extracted are held.SELECTED DRAWING: Figure 6

Description

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

  Flow cytometry refers to a method in which an antibody or the like labeled with a fluorescent dye is bound to a target cell, and particles are analyzed and separated by the fluorescence or scattered light. Under the present circumstances, the method of isolate | separating a specific cell (a microbe is included) from microparticles | fine-particles groups, such as a cell, is a cell sorting technique (nonpatent literature 1 grade | etc.,). Cell sorting technology is a very important method to understand 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, and first, the fluorescent dye labeled to the cells in the flow channel In accordance with the above, excitation light is irradiated, and the wavelength or intensity of fluorescence or scattered light emitted from each cell is analyzed to identify a desired cell. Then, a voltage is applied to the cells having specific properties identified by the analysis result such as the wavelength and intensity of the light to charge the cells, and discrimination, quantification and statistical analysis of the cells charged in the above using the deflection electrode Etc.

Prior literature

"Cell Engineering" separate volume "Flow cytometry free", supervision: Keimitsu Nakauchi (Univ. Of Tsukuba Medical School Immunology), Shujunsha, published July 1, 1999, page 3-23

  As described above, the conventional cell sorting technology is a technology for sequentially analyzing and collecting single cells flowing along the flow path. More specifically, it is a technique of analyzing the absolute value of the signal (mainly fluorescence or scattered light) emitted from the cell at the moment of passing through the detection unit. Therefore, it is unsuitable for conducting analysis on many items for many cells. Furthermore, the technology is also a technology that is also concerned about the occurrence of false positives and false negatives. On the other hand, there is a microarray technology as a method for reducing the frequency of false positives and false negatives and analyzing a large number of items for a large number of cells. However, in the art, when cells desired to be obtained are found, in order to obtain the cells by the method of inserting the micromanipulator into the microwell containing the cells, it is possible to automate acquisition of desired cells. Is unsuitable. Therefore, it is an object of the present invention to provide an object (for example, cell) acquisition technique capable of analyzing a plurality of subjects (for example, cells) simultaneously while reducing the frequency of false positives and false negatives and capable of automation. I assume.

The invention is as follows:
[1] a channel having a plurality of microwells,
An introduction path which can introduce a liquid containing a plurality of analytes (for example, test cells) into the flow path;
An information acquisition unit that acquires information from a plurality of subjects (for example, test cells) stored in the plurality of microwells;
A selective extraction unit capable of selectively extracting an analyte (for example, a test cell) in one microwell from the microwell based on the information acquired by the information acquisition unit;
A cell sorter or a flow cytometer having a subject (for example, a test cell) recovery unit capable of recovering a subject (for example, a test cell) selectively removed by the selective removal means.
The plurality of microwells can accommodate a subject (eg, a test cell) and a degradable gel (eg, a photodegradable gel);
For the degradable gel (e.g., photodegradable gel) in the microwell in which the subject (e.g., test cell) to be taken out is retained, the selective extraction means is the degradable gel (e.g., photodegradable gel). A cell sorter or a flow cytometer configured to be a decomposition processing unit (for example, decomposition light irradiation unit) that is subjected to decomposition processing (for example, capable of irradiating light of a wavelength that the photolytic gel decomposes).
[2] The cell sorter according to the above [1] or the cell sorter according to the above [1], wherein the flow channel is planar, and the plurality of microwells are provided in a lower wall (of upper and lower walls) constituting the flow channel. Flow cytometer.
[3] The information acquisition unit
The information from the plurality of microwells may be acquired for each microwell, for at least two of the plurality of microwells at one time, or for all the plurality of microwells at one time. Cell sorter or flow cytometer of 1] or [2].
[4] The information acquisition unit
An information source wave irradiation unit capable of irradiating an information source wave serving as a source of the information to be acquired to the plurality of microwells;
When the information source wave irradiator irradiates the information source wave to the plurality of microwells, the information source wave irradiator further includes an information receiver that receives the information that may be generated from the plurality of microwells. Cell sorter or flow cytometer of [3].
[5] In the first aspect, the information source wave irradiator can irradiate the information source wave for each microwell; in the second aspect, two or more of the plurality of microwells at one time Or, in a third embodiment, all of the plurality of microwells can be irradiated at one time; in the case of the first and second embodiments, all of the plurality of microwells are scanned The cell sorter or flow cytometer of the above-mentioned [4], which can be irradiated.
[6] The cell sorter or flow cytometer according to [4] or [5], wherein the information source wave irradiator can output a plurality of types of information source waves according to the number of analysis items.
[7] The information source wave irradiated from the information source wave irradiating unit is light, the degradable gel is a photodegradable gel, and the decomposable processing unit is a process of decomposing the photodegradable gel When it is a light irradiation part which can irradiate the light of a wavelength, the cell sorter or flow cytometer of said [4]-[6] whose wavelength of the said light differs from the wavelength of the light which the said photolytic gel decomposes | disassembles.
[8] The cell sorter or flow cytometer
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 gel is contained in a degradable gel (e.g. photo-degradable gel) in the microwell containing the subject (e.g. test cell) desired to be obtained. The cell sorter or the flow according to the above [1] to [7], further comprising selective extraction control means for controlling the selective extraction means to perform decomposition processing (for example, irradiation with light of a wavelength that the photolytic gel decomposes). Cytometer.
[9] A microfluidic device provided with the flow path, which is for the cell sorter or flow cytometer of the above [1] to [8].
[10] A liquid (for example, a liquid containing the following first component) containing a material capable of forming the degradable gel, which is for the cell sorter or flow cytometer of the above [1] to [8], A solution containing at least a component).
[11] A filling step of flowing a liquid containing a subject (for example, a test cell) in a channel having a plurality of microwells, and filling the microwell with the subject (for example, a test cell);
An analysis step of analyzing the sample (for example, a test cell) filled in the microwell after the filling step;
A subject (for example, cell) recovery method including an acquisition step of taking out the subject (for example, a test cell) after the analysis step,
The liquid contains a first component and a second component different from the first component,
The first component has a first part in which the basic skeleton is a biocompatible polymer and capable of binding to the second component,
The second component has a second part whose basic skeleton is a biocompatible polymer and capable of binding to the first component,
In the analysis step, a gel in which the first component and the second component are crosslinked due to the bond formation between the first portion and the second portion is formed in the microwell. ,
The first component and / or the second component further comprises a degradable moiety (eg, a photodegradable moiety),
In the acquiring step, the degradable portion (e.g., photodegradable portion) is decomposed by decomposing treatment (e.g., light irradiation) to decompose the gel and take out the subject (e.g., test cell) , A method of collecting a subject (eg, cell).
[12] The method of collecting a subject (eg, cell) according to [9], wherein the first part is an azide and the second part is an alkyne.

  According to the present invention, it is possible to provide an object (for example, cell) acquisition technique capable of analyzing a plurality of subjects (for example, cells) simultaneously while reducing the frequency of false positives and false negatives and capable of automation. .

FIG. 1 is a cross-sectional view of a microfluidic device A. FIG. FIG. 2 is a top view of the microfluidic device A. FIG. FIG. 3 is an exploded view of microfluidic device A. FIG. FIG. 4 shows an example of the chemical structural formula of the bonding part during gelation between the first component and the second component, which are gelation components, and the decomposition part when irradiated with light of a wavelength at which the gel decomposes. Is an example of the chemical structural formula of FIG. 5 is a view showing that a test cell is immobilized in a microwell by using the microfluidic device and the first and second liquids. FIG. 6 is a conceptual perspective view in the analysis process of the cell sorter or flow cytometer which is an example of the present invention. FIG. 7 is (a) a schematic perspective view and (b) an operation view in the cell acquisition step of the cell sorter or flow cytometer which is an example of the present invention. FIG. 8 is a schematic view showing the overall configuration of the cell sorter 1 in the cell sorter 1 which is 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 cell analysis control processing in the cell sorter 1 which is an example of the present invention. FIG. 11 is a flowchart of cell acquisition control processing in the cell sorter 1 which is an example of the present invention. FIG. 12 is a flowchart according to a modification 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 fluorescent protein-expressing Ba / F 3 cells immobilized in a microwell in Example 2. FIG. 16 is a fluorescence microscopic image of fluorescent protein-expressing Ba / F3 cells immobilized in a microwell in Example 3. FIG. 17 is a fluorescence microscopic image of fluorescent protein-expressing Ba / F3 cells immobilized in a microwell in Example 4. FIG. 18 shows a conventional cell sorting technique.

Hereinafter, the present invention will be described more specifically. Here, the “analyte” 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, a “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 performing a predetermined treatment, and a gel that can be decomposed by energy (for example, light, sound or heat), a decomposing agent Mention may be made of gels which can be degraded by the addition. However, in the following, as a "degradable gel", a "photodegradable gel" which is a preferred embodiment of the present invention will be described by way of example. The description will be made according to the following items. Moreover, in the following description, even when it is described as "cell sorter" or "flow cytometer", it shall include what was read as "flow cytometer" or "cell sorter".
(1. Cell sorting method)
1-1. Filling step 1-1-1. Structure of microfluidic device 1-1-2. First liquid to flow into the microfluidic device 1-1-2-1. First component and second component · Basic skeleton · First part / second part · Photodegradable part 1-1-3. Second liquid to be flowed into the microfluidic device 1-1-4. Process 1-2. Detection step 1-3. Analysis process 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 unit for gel decomposition

<< 1. Cell sorting method
The cell sorting method according to the present invention is
Filling the test cell-containing solution in a flow channel having a plurality of microwells, and filling the microwells with the test cells;
After the filling step, the microwell is irradiated with an information source wave (for example, laser light), and a signal detection step of detecting a signal caused by the irradiation;
Analyzing the test cells packed in the microwell based on the signal in the signal detection step;
And an acquisition step of taking out the test cell after the analysis step. Each step will be described in detail below.

<1-1. Filling process>
In the filling step, as described above, the first solution containing the test cells is flowed in the channel (microfluidic device) having the microwells, and the test cells are filled in the microwells, and then the The second solution is a step of removing the first solution (the first solution containing cells that did not enter the microwell or the solution containing a partially gelled solution) outside the microwell. The filling step will be described in detail with reference to FIGS. 1 to 5.

{1-1-1. Structure of microfluidic device}
An example of the microfluidic device A is shown in FIGS. 1 to 3. 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. And as shown in FIG.1 and FIG.3, the said microfluidic device A is the insertion opening Aa for introduce | transducing a liquid in the said gap (Introduction for the liquid insertion inserted in the insertion opening Aa. A port A ₁ ) and an opening A-b for discharging the fluid in the gap (a port A ₂ capable of sucking in and discharging the liquid discharged from the port A- _b ); ing. Then, as shown in FIGS. 1 to 3, a large number of microwells A-3 are provided on the lower surface of the microfluidic device A. Here, although the diameters of 20 μm, 30 μm and 40 μm are illustrated as the diameters of the microwells in FIG. 3, the present invention is 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 a common mammalian cell, it is more preferably 15 to 30 μm. If it is smaller than the preferred diameter, the cell filling rate is reduced, and if it is larger, a plurality of cells are filled in a single well, resulting in a decrease in single cell filling rate. Also, the well density in the microfluidic device A is not particularly limited, but is 100 to 10,000 / mm ² from the viewpoint of performing single cell capture with high efficiency. In the case of general mammalian cells, more preferably, it is 500 to 2,000 cells / mm ² . If it is smaller than the preferred well density, the number of cells that can be processed in one analysis decreases. Further, the upper limit of the well density is determined by the density when the above-mentioned preferred diameters are arranged in the closest packing. Furthermore, the well spacing is also not particularly limited, but preferably 2 μm or more. The microfluidic device includes at least a channel through which a liquid can pass, a plurality of microwells provided on the wall of the channel, an inlet for injecting a fluid into the channel, and the channel And an outlet for discharging fluid from the

  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, etc. .

  Further, as shown in FIG. 1 and FIG. 3, in the microfluidic device A, the upper surface A-1 and the lower surface A-2 are respectively constituted by different members, and the microdevice A intervenes in the upper surface A-1 and the lower surface A-2. Although the side wall A-4 which forms a channel by performing is provided, this is an example to the last, and upper surface A-1, lower surface A-2, and side wall A-4 are integrally formed, etc. There is no limitation as long as it can be used for the purpose of Moreover, in this example, although upper surface A-1 and lower surface A-2 of the microfluidic device A are made into planar structure, it is not limited to this, but fine unevenness 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 solution becomes smooth, and it becomes possible to more efficiently fill the microwell with the first solution and the test cells. Further, in this case, it is preferable that the microwell is provided on the lower wall of the upper and lower walls (that is, the lower surface A-2 in this example) constituting the flow path 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. The microwell may be provided on the upper wall (upper surface A-1), the side wall A-4, or the like that constitutes the flow channel, depending on the application.

{1-1-2. First fluid to flow into the microfluidic device}
The first liquid to be flowed into the microfluidic device is a liquid containing a plurality of test cells, and contains a first component and a second component different from the first component. Here, the first component and the second component react with time and gelate. The first component and the second component are present in the state of being dissolved in a liquid (for example, a liquid in which cells can survive, such as physiological saline or liquid medium). Hereinafter, each component contained in a 1st liquid is explained in full detail.

(1-1-2-1. First component and second component)
The first component and the second component are, as mentioned above, gelling materials. Here, the first component has a first part in which the basic skeleton is a biocompatible polymer and capable of binding to the second component. Also, the second component has a second part in which the basic skeleton is a biocompatible polymer and can be bound to the first component. Furthermore, the first component and / or the second component further comprises a photodegradable moiety. Hereinafter, the “basic skeleton”, the “first and second portions”, and the “photodegradable portion” of the first component and the second component will be described in detail.

-Basic skeleton The biocompatible polymer constituting the basic skeleton is not particularly limited, and a carbohydrate base polymer (methylcellulose, carboxymethylcellulose, hydroxymethylcellulose, hydroxypropylcellulose, hydroxyethylcellulose, ethylcellulose, dextrin, cyclodextrin, alginate, hyaluronic acid and Chitosan etc.) Protein based polymers (gelatin, collagen and glycol protein etc.) Hydroxy acid polyester (biodegradable polylactide-co glycolide (PLGA), polylactic acid (PLA), polyglycolide, polyhydroxybutyric acid, polycaprolactone, poly Valerolactone, polyphosphazene and polyorthoester etc.); albumin; polyanhydrides; polyethylene glycol Polyvinyl polyhydroxyalkyl methacrylates; pyrrolidone, polyvinyl alcohol. Among these, polyethylene glycol is preferred. In particular, multi-arm PEG (for example, 2-arm, 4-arm, 8-arm) is suitable. Moreover, the weight average molecular weight of PEG is preferably 500 to 100,000, and more preferably 2,000 to 40,000. Such PEGs are excellent in that they can recover cells in a form that does not impair their inherent functions (in other words, they can recover cells while they survive) because they have less influence on the cells. Here, the weight average molecular weight is a value measured by MALDI-TOF-MS.

-The first part / the second part The second 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 base of the second component which is the other side constituting the gel It can be linked to the second part linked to the backbone (or the first part linked to the basic backbone of the first component). As a combination of such a first part and a second part, for example, an azide group and an alkyne group (cycloaddition reaction), which is a combination of reactive groups that chemically form a bond in a liquid, and an azide group Dibenzocyclooctin group (cyclization addition reaction), thiol group and maleimide group (Michael addition reaction), thiol group and iodoacetamide group, thiol group and vinyl sulfone group, aldehyde group and hydrazine group, ketone group and hydrazine group, aldehyde group And aminooxy groups, ketone groups and aminooxy groups, combinations of proteins and ligands that form chemical bonds or strong interactions, biotin groups and streptavidin, maltosyl groups and maltose binding proteins, glutathionyl groups and glutathione S-transferase, HaloTag® ligand and H loTag (registered trademark) protein, guanylylmethylphenyl group and SNAP-tag (registered trademark), sitocinylmethylphenyl group and CLIP-tag, Strep-tag (registered trademark) and Strep-tactin (registered trademark), antigen and the like And antibodies. In addition, visible light activated polymerization which has a wavelength different from that of the photodegradable group using any of acrylic monomers, methacrylic groups, vinyl groups and epoxy groups which are polymerizable monomers as the first part and the second part It may be one that can be bonded by polymerization using any of the initiators Eosin Y, Rose Bengal, Camphor-quinone, and Erythrosin. Among 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 and second parts to the biocompatible polymer can be performed by a known method.

-Photolytic moiety The photolytic moiety may be present in one or both of the first component and the second component. Here, the photocleavable moiety refers to any group capable of leaving upon irradiation with light, and examples thereof include a nitrobenzyl group, a nitrophenylethyl ester group (NPE), a dimethoxynitrobenzyl ester group (DMNB), and a 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 ' -Methoxybenzoynyloxycarbonyl (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-nitroindinyl oxycarbonyl (BNIOC) group, etc. be able to. Among these, a group having a 2-nitrobenzyl derivative skeleton is preferable because it does not photolyze at room illumination level such as ordinary fluorescent lamps and incandescent lamps.

  Here, FIG. 4 shows an example of the chemical structural formula of the bonding portion at the time of gelation between the first component and the second component, which are gelation components, and light irradiation (light of the wavelength at which the gel decomposes) ) Is an example of the chemical structural formula of the decomposition part at the time of Here, as the first component, 4-arm PEG-PL-azide (4-arm PEG is linked to a photocleavable 2-nitrobenzyl derivative skeleton, and the azide is further bound to the 2-nitrobenzyl derivative skeleton And the second component is 4-arm PEG-DBCO (4-arm PEG attached to dibenzylcyclooctin). After these are added and mixed into the solution, crosslinks are formed between the azide and DBCO.

  The first liquid contains the first component and the second component at the time of use as described above. However, before use, that is, when transporting or storing the first solution, the composition preferably contains the first component to prevent the gelation reaction between the first component and the second component before use. It is an aspect of a kit consisting of at least two liquids of a liquid and a liquid containing a second component.

{1-1-3. Second fluid to flow into the microfluidic device}
After flowing the first liquid containing the test cells and the gelling material into the gap of the microfluidic device A, the first liquid is removed from the microwell by flowing the second liquid. The second liquid to be used at this time is not particularly limited, but a component incompatible with the first liquid, such as oil, is suitable.

{1-1-4. process}
FIG. 5 is a view showing that a test cell is immobilized in a microwell by using the above device and liquid. As described with reference to FIG. 5, the first solution (gel solution) is injected into the inside from the inlet of the microfluidic device ((a) in the figure). The operation is repeated a plurality of times (five to ten times in the example of the figure) ((b) in the figure). By this operation, cells can be efficiently filled into the microwells. However, the injection operation may be performed once. After that, the second liquid (oil) is injected into the inside from the inlet of the microfluidic device, and the excess first liquid present inside is discharged out of the microfluidic device ((c) in the figure). After that, incubation is performed under predetermined conditions, the gel material (first component and second component) in the microwell is gelled, and the cells housed in the microwell are immobilized.

  In the present embodiment, the first solution (gel solution) is filled in the microwell before gelation, but this is not a limitation. More specifically, the first solution (gel solution) in a state in which the first solution (gel solution) around the test cells is gelled, and the entire first solution is not gelled. It is preferable to inject into the inside from the inlet of the microfluidic device. By such a process, the gel around the test cell functions as a protective material, and the test cell can be filled in the microwell in such a manner that the function originally possessed by the cell is not impaired.

<1-2. Detection process>
Signal detection from a test cell fixed by gel in the microwell can be made similar to the detection method used in conventionally known flow cytometry and cell sorter. For example, by irradiating a wave serving as an information source to a target and observing the response, it is possible to acquire information of a detection target. As a more specific example, the detection is obtained by irradiating the microwell to be detected with a laser beam (for example, single laser or dual laser such as argon, diode, die, helium neon, etc.) and obtaining the irradiation The measurement can be carried out by measuring signals {forward scattered light (FSC) or side scattered light (SSC), and various fluorescences of the fluorescently labeled cells when target cells are previously labeled with a fluorescent substance}. In addition, it does not specifically limit as a wave used as such an information source, The electromagnetic waves which have appropriate wavelengths, such as a gamma ray-a microwave, a sound wave, etc. can be illustrated.

<1-3. Analysis process>
The analysis may be similar to detection methods used in conventionally known flow cytometry and cell sorter. For example, through analysis based on the signals (data) obtained in the detection step, it is possible to analyze cell size, complexity of the internal structure of cells, and the like. In particular, according to the present invention, it is also possible to conduct more detailed analysis such as imaging of cells (for example, analysis of dynamics of cell membrane molecules, analysis of cell chromosomes). One feature of the present invention is that since test cells are immobilized in the microwell, analysis can be performed on a plurality of items at the same time.

<1-4. Acquisition process>
The acquisition is carried out by irradiating the microwell containing the test cells to be acquired with light (light of a wavelength that causes the photolytic gel in which the test cells are embedded in the microwell to be decomposed). . The photolysable gel in the microwell irradiated with the light is decomposed, and the fixation of the test cells in the microwell is released (drops below the microwell). Thereafter, the test cell is discharged together with the liquid (for example, as liquid to be released into the flow path in such an acquisition process, to the cells by flowing the liquid (for example, physiological saline) into the flow path. A liquid with little damage, etc., such as 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 different types of desired cells can be selectively obtained from the same array.

  In addition, although the analysis was performed immediately after embedding cells with gel in a microwell in the above, and the aspect which acquires a desired cell was illustrated, it is not limited to this. For example, after embedding cells in a microwell with a gel, the culture solution may be poured, and the cells may be cultured in the wells (eg, cultured for a long time), and the cells after the culture may be analyzed. .

<< 2. Cell sorter »
Next, the cell sorter or flow cytometer which is an example of the present invention will be described in detail with reference to FIGS. 6 and 7. The basic configuration of the cell sorter or the flow cytometer is the same as that of a conventionally known one. Therefore, points different from the conventional one will be mainly described.

The cell sorter or flow cytometer 1 which is an example of the present invention is
A channel (microfluidic device) 1-1 having a plurality of microwells 1-1-a,
A laser beam irradiation unit 1-2 capable of irradiating a laser beam to one microwell of the plurality of microwells 1-1-a;
When the laser beam irradiation unit 1-2 irradiates a laser beam to the one microwell, a signal detection unit 1-3 that detects a signal generated due to the irradiation;
An analysis unit (not shown) for analyzing a test cell in the one microwell based on the signal from the signal detection unit 1-3;
The laser beam irradiation unit 1-2, the signal detection unit 1-3, and the analysis unit (not shown) perform the irradiation, the irradiation, and the irradiation on all of the plurality of micro wells 1-1-a. A cell sorter 1 configured to be capable of performing detection and the analysis, wherein
The microwell 1-1-a can accommodate a test cell and a photodegradable gel,
The cell sorter 1 is
A light source for gel decomposition, which is light for decomposing the photolytic gel after the analysis by the analysis unit (not shown), and emits light of a wavelength different from that of the laser beam
Furthermore, it has. Each part will be described in detail below.

<2-1. Microfluidic Device>
Since the microfluidic device has been described above, the description thereof is omitted (the microfluidic device A shown in FIGS. 1 to 3 and the microfluidic device 1-1 shown in FIGS. , Has the same function). In addition, light irradiation (light irradiation from the laser beam irradiation part 1-2 and / or the light irradiation part 1-4 for gel decomposition) to the microwell 1-1-a in the microfluidic device 1-1 or signal detection ( 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 section>
The laser beam irradiation unit 1-2 is configured to be able to independently irradiate each of the plurality of microwells 1-1-a. For example, in the example of FIG. 6, the microwells 1-1-a are configured to be movable (back and forth and left and right) directly below (or immediately above) each of the plurality of microwells 1-1-a (so-called scanning type). In this case, the signal detection unit 1-3 described later is also configured to be movable (back and forth and left and right) following the movement of the laser beam irradiation unit 1-2 (see FIGS. 6A and 6B). ). In addition, the method of light-irradiating independently to each of several micro wells 1-1-a is not restricted to this, For example, irradiation is performed by a swing under the condition where the root of laser beam irradiation part 1-2 is being fixed. A method of changing the position of the micro well 1-1-a, a method of providing the laser beam irradiation unit 1-2 by the number of the plurality of micro wells 1-1-a, a plurality of micro wells 1-1-a at one time Method of irradiation (for example, a method of arranging a laser beam irradiation unit in a line to form a linear light source, or a certain range is defined as one section, and a plurality of micro wells in the one section can be irradiated with light) And the like), a method of irradiating all the plurality of microwells at one time, and the like (such as using a light source capable of irradiating all the microwells with light). In the case where a plurality of analyzes are performed simultaneously, a plurality of laser beam irradiation units may be provided in correspondence with the types of analysis. In addition, the laser beam irradiation unit may change the type of the detection wave, and a plurality of analyzes may be simultaneously performed. In this example, the light is irradiated from directly below (or immediately above) the microwell 1-1-a, but light is obliquely emitted from below (or obliquely above) the microwell 1-1-a. Irradiation may be performed.

<2-3. Signal detection unit>
The signal detection unit 1-3 detects a signal emitted when the laser is irradiated to the test cell by the laser beam irradiation unit 1-2. As in the case of the laser beam irradiation unit 1-2, since it is necessary to detect the signal of each of the plurality of micro wells 1-1-a, the movement of the laser beam irradiation unit 1-2 is performed as described above in the example of FIG. Are configured to be movable (back and forth and left and right) (see FIGS. 6A and 6B). The method of detecting the signal of each of the plurality of microwells 1-1-a is not limited to this. For example, a method of providing the signal detection unit 1-3 by the number of the plurality of microwells 1-1-a, a plurality of A method of detecting a signal from a microwell (for example, a method of arranging a plurality of signal detection units 1-3 in a line, or a certain range is one section, and signals from a plurality of microwells in the one section are It may be a method of making it detectable or a method of detecting signals from all the plurality of microwells at one time. For example, when the signal detection unit is a microscope, the configuration can be changed as appropriate, such as imaging one microwell or imaging a plurality of microwells simultaneously. Further, 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 in a form corresponding to one microwell And install). In this example, the signal detection unit 1-3 is shown to be on the opposite side of the laser beam irradiation unit 1-2 via 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 according to 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 decomposition light irradiation unit 1-4 is configured to be able to independently irradiate each of the plurality of microwells 1-1-a. In addition, in FIG. 7, although it describes as the same thing as the laser beam irradiation part 1-2 from a viewpoint of simplification of drawing, the light of the laser beam irradiation part 1-2 and the light irradiation part 1-4 for gel decomposition are 1-4. The light source is usually a different light source because it has a different wavelength. However, when it is a light source which can change the wavelength of the light to irradiate, it is not necessary to divide the laser beam irradiation part 1-2 and the light irradiation part 1-4 for gel decomposition | disassembly. Furthermore, it may be a light source in which a plurality of light sources (the laser beam irradiation unit 1-2 and the gel decomposition light irradiation unit 1-4) are unitized. Here, as in the case of the laser beam irradiation unit 1-2, the gel decomposition light irradiation unit 1-4 is also movable (or directly above) each of the plurality of micro wells 1-1-a in the example of FIG. Front and rear, left and right). As in the case of the laser beam irradiation unit 1-2 described above, the method of irradiating light independently to each of the plurality of micro wells 1-1-a is not limited to this. For example, the light irradiation unit 1-4 for gel decomposition is used. A method of changing the position of the microwell 1-1-a to be irradiated by swinging under the situation where the root of the gel is fixed, or the light decomposition unit 1-4 for gel decomposition is used for the plurality of microwells 1-1-a. It may be a method of providing only the number.

  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, multiple cells are simultaneously imaged (microscopic analysis) with a confocal laser microscope to sort the cells. After that, in the well containing the cells of interest, the region of interest (CLSM) mode is used in the well and the light of the light decomposition wavelength (for example, light of 405 nm) is irradiated to that portion to decompose the gel, etc. do it.

  In the cell sorter or flow cytometer of this example, various liquids can be fed from the introduction port 1-1-b of the microfluidic device 1, and the decomposed gel or the like is discharged to the discharge port 1-1 of the microfluidic device 1. The box 1-6 is provided at the discharge port 1-1-c for recovering cells contained in the discharged liquid and gel (such liquid transfer, Evacuation, cell recovery, etc. may be performed by appropriate means as needed).

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

  In the cell sorter 1 of this example, the laser beam irradiation unit 1-2 (laser irradiation unit 1-2) capable of irradiating a laser beam corresponding to one mic well shown in FIGS. 6 and 7, a laser irradiation unit A signal detection unit 1-3 capable of detecting a signal derived from the light irradiation 1-2, and a gel decomposition light irradiation unit 1-4 capable of light irradiation corresponding to one microphone well It is configured to be movable (front and rear, left and right).

  First, FIG. 8 is a schematic view showing the entire 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, the liquid phase portion 2, the cell recovery portion 3, and the display portion 4.

  Here, the liquid transfer unit 2 is a device capable of filling various liquids (the first liquid, the second liquid, and a liquid such as physiological saline described above) in the microfluidic device 1-1, and in the device. By changing the flow path, it is possible to select and switch the liquid to be fed appropriately. The cell recovery unit 3 is an apparatus (for example, the box 1-6 described above) capable of recovering cells discharged from the microwell 1-1-a. The display unit 4 is an apparatus capable of displaying various data (for example, cell information and the like described later) of the processing unit 100 as an image. The other configurations are the same as described above and thus will not be described.

  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 that can control an apparatus related to an analysis process / detection process, a gel decomposition apparatus control unit 120 that can control an apparatus related to an acquisition process, and A liquid transfer control unit 130 capable of controlling the liquid unit 2 and a cell collection control unit 140 capable of controlling cell acquisition behavior of the cell collection unit 3 (for example, opening and closing of an opening for introducing cells, position of the opening, etc.) , An information storage unit 150, a cell information determination unit 160 capable of performing various determinations based on cell information stored in the information storage unit 150, display control capable of controlling an image output of the display unit 4 and the like And a unit 170. As shown in this figure, in this example, a detection device having a laser irradiation unit 1-2 and a signal detection unit 1-3, a gel decomposition apparatus having a gel decomposition light irradiation unit 1-4, and The liquid unit 2, the cell collection unit 3, and the display device 4 are electrically connected to the processing unit 100.

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

  In addition, the gel decomposition apparatus control unit 120 includes a gel decomposition light irradiation control unit 121-1 capable of controlling a light irradiation mode (irradiation time, irradiation intensity, etc.) in the gel decomposition light irradiation unit 1-4, and for gel decomposition. It has a gel decomposition 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 according to the cell analysis control process (step 1000) in the cell sorter 1.

  First, in a situation 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 or not cell analysis control has been started. If Yes in step 1001, in step 1002, the fluid delivery control unit 130 delivers the cell-containing liquid (first fluid) from the fluid delivery 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 fluid delivery control unit 130 delivers the oil from the fluid delivery unit 2 into the microfluidic device 1-1, and removes the excess cell-containing fluid in the microfluidic device 1-1. (Note that the liquid fed in step 1004 is not limited to oil, and may be the above-mentioned second liquid). 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 under the situation where it is linked with the position information of 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 n position. Next, in step 1010, the laser irradiation control unit 111-1 causes the laser irradiation unit 1-2 to irradiate the detection well to the microwell present at the position n (in this case, the signal detection unit 1-). 3 detects a signal obtained when the detection laser is irradiated). Next, in step 1012, based on the signal detected by the signal detection unit 1-3, the processing unit 100 temporarily stores cell information related to cells present in the microwell irradiated with the detection laser in the information storage unit 150. In this embodiment, even if there is no cell in the well, it is temporarily stored as cell information, but if there is no cell in the well, the cell information on the corresponding microwell will be stored. May not be stored). Next, in step 1014, the processing unit 100 determines whether the irradiation of the detection laser to all the microwells has ended (whether n has reached to fin). In the case of Yes in step 1014, the process proceeds to step 1018. On the other hand, in the case of No at step 1014, the processing unit 100 adds 1 to n at step 1016, and proceeds to step 1008. Next, in step 1018, the cell information determination unit 160 refers to all temporarily stored cell information {in this example, a 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 a desired cell is present in the microwell. If Yes in step 1020, the processing unit 100 temporarily stores the position information of the microwell in which the desired cell is present in the information storage unit 150, and shifts to step 2000.

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

  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, etc., steps 1006 to 2000 You may repeat to.

  Here, in the present example, the variable n and positional information of each microwell in the microfluidic device 1-1 are linked, but the positional information of the microwell may be positional information of the microwell itself The surface on which the microwells are provided may be divided appropriately (grouped), and the information on the division may be used, and it is not limited at all. Further, such microwell position information may be previously incorporated in the processing unit 100, or the position of the microwell may be automatically determined by an optical method or the like.

  In addition, in the case of movement of the laser irradiation part 1-2, after completion | finish of the analysis with respect to one microwell, the laser irradiation part 1-2 moves to the following microwell (a structure of the laser irradiation part 1-2) It is not limited to the configuration in which stop and move are repeated. For example, the laser irradiator 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, even if the position of the microwell does not exist, the position at which the desired cell information is obtained can be read as the position information of the microwell, and thus presetting of the position information of the microwell, etc. The analysis start condition and end condition may be changed as appropriate).

  Further, in this example, it is assumed that one laser irradiation is performed on one microwell, but as described above, it is possible to analyze a plurality of microwells by one laser irradiation. May be In particular, in the case where each microwell has a plurality of laser irradiators corresponding to each other, or in a configuration where analysis can be performed on all the plurality of microwells by one laser irradiation, etc. The laser irradiation unit 1-2 may not move because it is possible to acquire all cell information while the stop is stopped. Further, in this example, the laser irradiator 1-2 itself is moved, but as described above, the laser irradiator 1-2 may be oscillated, or the irradiation angle may be changed by a reflection plate. The configuration may be such that the microwell to be analyzed is changed.

  In addition, as described above, the detection laser irradiated from the laser irradiation unit 1-2 used in the analysis step is not limited to the laser light, and has a function capable of irradiating an appropriate information source wave capable of acquiring cell information. There is no limitation whatsoever, as long as it exists. Furthermore, as described above, the laser irradiation unit 1-2 is a configuration capable of oscillating a plurality of information source waves (that is, a configuration capable of changing the oscillation wavelength, frequency, etc., and a configuration including a plurality of oscillation terminals). A plurality of laser irradiators 1-2 may be provided depending on the type of information source wave to be oscillated.

  Moreover, 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 can be performed. For example, in a simple manner, cell information related to all the plurality of microwells may be detected by one fixed signal detection unit 1-3 (in this case, the signal detection unit 1-3). Control related to the movement of Moreover, when analyzing with a plurality of information source waves, the signal detection unit 1-3 may be configured to have one signal detection unit 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), in order to gelate the component in the cell-containing liquid (first liquid) after step 1004, the processing of the latter stage may be made to stand by (or , And may be a treatment that promotes gelation). In addition, after preparing a plurality of microfluidic devices 1-1 and performing the processing according to step 1002 and step 1004 on a certain microfluidic device 1-1, successively, on another microfluidic device 1-1 The processing according to step 1002 and step 1004 may be configured to be executable. In other words, the processing according to step 1002 and step 1004 can be performed on another microfluidic device 1-1 during the waiting time required for gelation of the component in the cell-containing liquid in one microfluidic device 1-1 It may be In this case, the components in the cell-containing liquid in the microfluidic device 1-1 may be further configured to shift to the next process sequentially from the gelled ones.

  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 out position information of a predetermined microwell determined to have a desired cell. Next, in step 2004, the processing unit 100 performs an initial setting process of the gel decomposition light irradiation unit 1-4 (a variable m is linked to position information of a predetermined microwell in the microfluidic device 1-1. Under the circumstances, substitute 1 for m). In the present example, among the position information of the predetermined well read out in step 2002, the position on the most upstream side of the microfluidic device 1-1 is the initial position (m = 1). Next, in step 2006, the gel decomposition light irradiation position control unit 121-2 moves the gel decomposition light irradiation unit 1-4 to the position of m. Next, in step 2008, the gel decomposition light irradiation control unit 121-1 causes the gel decomposition light irradiation unit 1-4 to irradiate the gel decomposition laser to the microwell present at the position of m. Next, in step 2010, the solution sending control unit 130 sends the solution (for example, physiological saline) from the solution sending unit 2 into the microfluidic device 1-1 as a solution sending process (in this case, the solution is decomposed). Gel and 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 liquid supplied, the gel and the cells contained in the gel that have been degraded are Collect in 3 Next, in step 2014, the processing unit 100 determines whether or not the irradiation of the gel decomposition laser has been performed on all predetermined microwells. In the case of Yes in step 2014, the cell acquisition control process 2000 ends. On the other hand, if No in step 2014, in step 2016, the processing unit 100 adds 1 to m, and proceeds to step 2006.

  Further, in this example, it is assumed that one microwell is irradiated with a single gel decomposition laser, but in the case of having a plurality of laser irradiation parts corresponding to each microwell, one laser, etc. The configuration may be such that gel decomposition can be performed on a plurality of microwells by irradiation. Further, in this example, the gel decomposition light irradiation unit 1-4 itself is moved, but as described above, the gel decomposition light irradiation unit 1-4 is swung or irradiated by the reflection plate. The microwell for performing gel degradation may be changed by changing the angle or the like (in this case, gel decomposition in all the wells becomes possible while the light irradiation part 1-4 for gel degradation is stopped). And the light irradiation part 1-4 for gel decomposition does not need to move).

  Further, in the present example, the cell analysis control process (step 1000) and the cell acquisition control process (step 2000) are continuous processes, but they may be independent processes.

  In this example, although an example of a system that automatically performs up to the detection step and the analysis step has been described, the present invention is not limited thereto, and a system in which manual processing intervenes in part of the system. It is also good. An example of such a system is described below.

  Next, FIG. 12 is a flowchart of a cell analysis control process (S1000-2) according to a modification 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, in the case of 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 selects desired cells 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 judged whether or not it exists. In the case of Yes in step 1020-2, the process proceeds to a cell acquisition control process (step 2000).

  In the present modification, processing may be interrupted between step 1019-2 and step 1020-2. Before step 1020-2, that is, while the processing is interrupted, the operator may, for example, observe the cell information displayed on the display device 4 (for example, the display), and whether or not the desired cell is present Make a decision. The selection information (whether or not there is a predetermined microwell in which a desired cell is present, the position of the predetermined microwell, etc.) related to the result of the determination is processed using the input terminal etc. , 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 the determination regarding the cell information), the processing unit 100 may not have 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 part of the mode (a mode in which the operator makes an appropriate determination and performs various processes based on the determination or the like). For example, the liquid-feeding process of the cell-containing liquid, the liquid-feeding process of oil, the liquid-feeding (for example, physiological saline) process, the cell acquisition, and the like may be appropriately performed manually).

  Hereinafter, the present invention will be more specifically described with reference to examples. The present invention is not limited to the examples.

Example 1
(Synthesis of gel material)
The gel material used in the present example was synthesized based on the outline and the synthetic scheme of FIG. Hereinafter, the 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 repeating number n of the raw material 4-arm PEG-amine is about 111.
・ Synthesis of compound 2
The reaction was performed in the dark. In a 50 mL branched flask, 1.15 g (10.0 mmol, 2 eq) and 1-Ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) 1.99 g (10.0 mmol, 2 eq) of N-Hydroxysuccinate (NHS) The reaction mixture was dried for 20 minutes, replaced with nitrogen, and then 18 mL of dry DMF was added. Stir at room temperature to dissolve NHS and EDC, then add 1.51 g (5.01 mmol, 1 eq) of 4- [4- (1-hydroxyethyl) -2-methoxy-5-nitrophenoxy] butyric acid (1), Further, 2 mL of dry DMF was added. After stirring at room temperature for 10.5 hours, completion of the reaction was confirmed, and the solution was concentrated by an evaporator. The concentrated solution was dark yellow-brown, pure water was added, and the precipitated pale yellow solid was collected by suction filtration. Vacuum drying overnight gave a pale yellow solid. The yield was 1.97 g, 103%. The reason why the yield exceeded 100% is considered to be that the solvent DMF could not be removed. The identification of the product was performed by ¹ H-NMR (CDCl ₃ ). ¹ H NMR (600 MHz, CDCl ₃ , TMS) δ = 7.59 (s, 1 H), 7.31 (s, 1 H), 5.57 (q, J = 5.9 Hz, 1 H), 4.18 (t, J = 6.2 Hz, 2 H ), 3.99 (s, 3 H), 2. 90 (t, J = 8.5 Hz, 2 H), 2. 86 (br s, 4 H), 2. 29 (quin, J = 6.2 Hz, 2 H), 1.56 (d, J = 6.4 Hz, 3H)
・ Synthesis of compound 3
The reaction was performed under a light-shielded, argon atmosphere. In a 50 mL flask, 1.11 g (2.80 mmol, 1 eq) of the compound 2 was put into a dry state and then placed under an argon atmosphere, 10 mL of dry DMF was added, and the mixture was stirred at room temperature. The solution was orange and clear. 0.700 g (7.00 mmol, 2.5 eq) of 3-azidopropyl-1-amine was added and allowed to react at room temperature for 18 hours, and the solution was evaporated to form an orange clear oil. This was vacuum dried overnight. It was diluted with 100 mL of ethyl acetate and washed three times with pure water. The organic phase was dried over MgSO ₄ and after filtration the solution was evaporated to give an orange clear oil. Drying under vacuum gave a brown solid. The yield was 0.97 g, 91%. The identification of the product was performed by ¹ H-NMR (CDCl ₃ ). ¹ H NMR (600 MHz, CDCl ₃ , TMS) δ = 7.56 (s, 1 H), 7.31 (s, 1 H), 5. 91 (brs, 1 H), 5.56 (q, J = 10.3 Hz, 1 H), 4.11 (t , J = 8.8 Hz, 2 H), 3.99 (s, 3 H), 3. 35 (dt, J = 10.3 Hz, 4 H), 2.42 (t, J = 10.3 Hz, 2 H), 2. 20 (quin, J = 8.8 Hz, 2 H ), 1.78 (quin, J = 10.3 Hz, 2H), 1.56 (d, J = 10.2 Hz, 3H)
・ Synthesis of compound 4
The reaction was performed under a light-shielded, 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 placed, dried up, and placed under an argon atmosphere. When 2 mL of dry CH ₂ Cl ₂ was added and stirred, it was an orange clear solution. To this was added 110 mL of dry triethylamine (d = 0.726 g / cm ³ , 0.80 mmol, 4.7 eq), and the mixture was stirred for 1.5 hours. As a result, the disappearance of the raw material compound 3 was confirmed. It was directly connected to a vacuum pump, and when triethylamine and CH ₂ Cl ₂ were distilled off, it became a yellowish green oil. The silica gel column chromatography was performed with hexane: ethyl acetate = 1: 1 → ethyl acetate 100%. The column diameter was 2 cm and the column length was 10 cm. The fractions containing the desired product were evaporated and vacuum dried to obtain a pale yellow solid. The yield was 46.3 mg, and the yield was 50%. The identification of the product was performed 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, 2 H), 7.62 (s, 1 H), 7. 35 (d, J = 9.1 Hz, 2 H), 7.12 (s, 1 H) ), 6.53 (quin, J = 6.4 Hz, 1 H), 5.82 (brs, 1 H), 4.14 (t, J = 6.2 Hz, 2 H), 4.01 (s, 3 H), 3.36 (m, 4 H), 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 performed under a light-shielded, argon atmosphere. In a 10-mL two-necked flask, 152 mg (7.5 mmol, 1 eq) of PTE-200PA (SUNBRIGHT, terminal NH ₂ four-branched PEG, Mw ~ 20000) and 46.3 mg (84.7 mmol, 11 eq) of compound 4 are placed and dried. 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, the disappearance of the raw material PTE-200PA was confirmed. The solution was dropped on a tube containing 30 mL of diethyl ether on an ice bath, and a pale yellow precipitate formed. The tube was centrifuged (4 ° C., 10 krpm, 10 min), the supernatant 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 centrifuge the supernatant to MiiliQ 1.8 L, molecular weight fraction 3500 The dialysis was performed for 3.5 days with the dialysis membrane. Lyophilization gave a white solid of the objective 4-arm PEG-PL-azide. The yield was 80 mg, and the yield was 49%. Identification of products and measurement of molecular weight 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. 150 mg (7.5 mmol, 1 eq) of PTE-200PA and 21.5 mg (52.5 mmol, 7 eq) of DBCO NHS-ester are put into a 15 mL two-necked flask and dried up for N ₂ substitution, then dry CH ₂ It was dissolved in 2 mL of Cl _{2 and} stirred at room temperature for 18 hours. It was confirmed that the raw material PTE-200PA disappeared, and the reaction solution was poured into two 50 mL centrifuge tubes containing 20 mL each of diethyl ether in an ice bath to cause ether precipitation, and a white precipitate was formed. The supernatant was decanted off by centrifugation at 10 krpm for 7 minutes at 4 ° C., left to dry at room temperature and then vacuum dried in a desiccator. Three mL each of the 50 mM Tris / HCl (pH = 8.0) buffer prepared was added to each tube, and 2 mL each of MilliQ was further added and suspended. The solution was centrifuged at 3 krpm for 3 minutes at 4 ° C. and dialyzed against MilliQ 2 L using a 3500 molecular weight fraction dialysis membrane. Water exchange took place after 19.5 and 22.5 hours. Lyophilization gave a white solid. The yield was 130 mg, and the yield was 83%. Identification of products and measurement of molecular weight 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 (d, J = 7.3 Hz, 4 H), 7.52 (d, J = 8.2 Hz, 4 H), 6.23 (brs, 4 H), 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 several calcein-stained Ba / F3 cells in physiological saline, add two types of gel materials (4-arm PEG-PL-azide and 4-arm PEG-DBCO) shown in FIG. I got a liquid. The cell density is 50,000,000 cells / mL, and the concentration of the gel material is 0.9 w / v%.
(Cell fixation to 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 multiple times. Then, it was incubated at 4 ° C. for 5 minutes to precipitate cells into the microwells. 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. Thereafter, the cells were incubated at 4 ° C. for 20 minutes to gel the cell-containing solution in the microwells. As a result, as shown in FIG. 14, it was confirmed that cells were accommodated in 64% of the microwells.
(Cell analysis fixed to microwell)
The fluorescence emitted by irradiating laser to calcein-stained Ba / F3 cells fixed to the microwell was measured to analyze the size, shape, etc. of the cells.
(Removal of cells immobilized in microwells)
After the cell analysis, a specific microwell to which the cells were fixed was irradiated with light having a wavelength of 405 nm to decompose the gel in which the cells were embedded. Thereafter, flushed with saline from the inlet A ₁ of the microfluidic device A. As a result, it was possible to selectively obtain the cells immobilized in the specific microwell.
Example 2
Green fluorescent protein-expressing Ba / F3 cells were immobilized in the same manner as in Example 1, except that the red fluorescent dye-modified photodegradable hydrogel was used. Thereafter, light (405 nm) was irradiated on specific cell groups to dissolve the gel in these microwells. Then, these cells were selected and obtained by flowing a liquid (buffer under physiological conditions). Here, FIG. 15a is an image (a superimposed image of a green fluorescence and red fluorescence image and a bright field image) of a cell immobilized with a photolytic hydrogel in a microwell, and a portion surrounded by a square is a light. It is a microwell irradiated with (405 nm). Also, FIG. 15 b is an image of cells immobilized with photodegradable hydrogel in the microwell after being washed with a liquid (buffer under physiological conditions) a given number of times (15 times in this example) after light irradiation. is there. As can be seen from FIG. 15b, in this example, a plurality of (seven in this example) cell groups could be obtained collectively.
Example 3
In the same manner as in Examples 1 and 2, except that green fluorescent and red fluorescent protein (EGFP and Kusabira-Orange) expressing Ba / F3 cells were examined. FIG. 16 is a fluorescence microscope image of fluorescent protein-expressing Ba / F3 cells immobilized in a microwell {a is a superimposed image (low magnification) of a green fluorescence image and a red fluorescence image, b is Superimposed images (high magnification) of green and red fluorescence images and bright field images}. Although it is unclear from the figure, there are microwells that emit only green fluorescence, microwells that emit only red fluorescence, and microwells that emit red fluorescence and green fluorescence. In addition, the reason that red fluorescence is also emitted from cells emitting mainly green is because light emitted as scattered light from the cells themselves was detected when the laser light emitting red fluorescence was irradiated. On the contrary, when the laser light which emits green fluorescence is emitted, green fluorescence is emitted only from the cells expressing EGFP, and green fluorescence is not emitted from the cells expressing Kusabila-Orange. Also, although there are a few microwells containing two cells, it is possible to identify such microwells by comparing the fluorescence image and the bright field image.
Example 4
Only the cells having red fluorescence (first step) are selectively removed from the fluorescent protein-expressing Ba / F3 cells immobilized in the microwell with the photocleavable hydrogel prepared in Example 3 and then , (Second step) Only cells having green fluorescence were selectively removed. Here, FIGS. 17 a to 17 d are images of the cells immobilized with the photolytic hydrogel in the microwell (superimposed images of green fluorescence and red fluorescence images and bright field images). Although it is unclear from the figure, there are microwells that emit only green fluorescence, microwells that emit only red fluorescence, and microwells that emit red fluorescence and green fluorescence. And FIG. 17 a is an image before selective extraction of the first-step red fluorescent cells (portion surrounded by squares = well in which red fluorescent cells are present), and FIG. 17 b is a first-step red fluorescent cell Image after selective extraction (square part = well where red fluorescent cells were present). Furthermore, FIG. 17 c is an image before selective extraction of the second-step green fluorescent cells (squared portion = well in which green fluorescent cells are present), and FIG. 17 d is a second-step green fluorescent cell Image after selective extraction (square part = well where green fluorescent cells were present).

Claims (12)

A channel having a plurality of microwells,
An introduction path which can introduce a liquid containing a plurality of analytes into the flow path;
An information acquisition unit that acquires information from a plurality of subjects stored in the plurality of microwells;
A selective extraction unit capable of selectively extracting an object in one microwell from the microwell based on the information acquired by the information acquisition unit;
A cell sorter or a flow cytometer having a subject recovery unit capable of recovering a subject selectively removed by the selective removal means;
The plurality of microwells can store an analyte and a degradable gel;
A cell sorter or a flow cytometer configured such that the selective extraction means is a decomposition processing unit that subjects the degradable gel in the microwell in which the analyte to be extracted is held to the decomposition of the degradable gel.   The cell sorter or flow cytometer according to claim 1, wherein the flow channel is planar, and the plurality of microwells are provided in a lower wall constituting the flow channel. The information acquisition unit
The information from the plurality of microwells may be acquired per microwell, at one time for two or more of the plurality of microwells, or at one time for all the plurality of microwells. The cell sorter or flow cytometer according to 1 or 2. The information acquisition unit
An information source wave irradiation unit capable of irradiating an information source wave serving as a source of the information to be acquired to the plurality of microwells;
4. The information receiving unit according to claim 1, further comprising: an information receiving unit that receives the information that may be generated from the plurality of microwells when the information source wave irradiation unit irradiates the plurality of microwells with the information source wave. The cell sorter or flow cytometer according to any one of the above.   In the first aspect, the information source wave irradiator can irradiate the information source wave for each microwell; in the second aspect, can irradiate two or more of the plurality of microwells at one time Or, in the third aspect, all the plurality of microwells can be irradiated at one time; in the case of the first aspect and the second aspect, all the plurality of microwells are scanned and irradiated The cell sorter or flow cytometer according to claim 4, which is obtained.   The cell sorter or flow cytometer according to claim 4 or 5, wherein the information source wave irradiation unit can output a plurality of information source waves according to the number of analysis items.   The information source wave irradiated from the information source wave irradiator is light, the degradable gel is a photo-degradable gel, and the decomposition processing part is a light of a wavelength that the photo-degradable gel decomposes The cell sorter or flow cytometer according to any one of claims 4 to 6, wherein when the light irradiator is capable of irradiating the light, the wavelength of the light is different from the wavelength of the light decomposed by the photodegradable gel. 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 in which the object to be obtained is stored is decomposed. The cell sorter or flow cytometer according to any one of claims 1 to 7, further comprising selective extraction control means for   A microfluidic device comprising the flow path, for the cell sorter or flow cytometer according to any one of claims 1 to 8.   A liquid containing the material capable of forming the degradable gel, which is for the cell sorter or the flow cytometer according to any one of claims 1 to 8. A step of flowing a solution containing an analyte into a channel having a plurality of microwells, and filling the microwell with the analyte;
An analysis step of analyzing the analyte filled in the microwell after the filling step;
A subject recovery method comprising: an acquisition step of taking out the subject after the analysis step;
The liquid contains a first component and a second component different from the first component,
The first component has a first part in which the basic skeleton is a biocompatible polymer and capable of binding to the second component,
The second component has a second part whose basic skeleton is a biocompatible polymer and capable of binding to the first component,
In the analysis step, a gel in which the first component and the second component are crosslinked due to the bond formation between the first portion and the second portion is formed in the microwell. ,
The first component and / or the second component further comprises a degradable portion,
In the acquisition step, a decomposition method is performed to decompose the degradable portion to decompose the gel and take out the analyte.   The method for collecting an analyte according to claim 11, wherein the first part is an azide and the second part is an alkyne.