Target cell preparation method and system

Abstract

To provide a technique for improving reaction efficiency, introducing an active agent uniformly into source cells, and producing homogeneous target cells.SOLUTION: According to one embodiment, a target cell preparation method includes forming a first water-in-oil droplet containing source cells by mixing an aqueous solution containing the source cells with an oil phase, forming a second water-in-oil droplet containing liposomes by mixing a liposomal aqueous solution encapsulating an active agent with an oil phase, fusing the formed first water-in-oil droplet with the second water-in-oil droplet, and introducing the active agent into the source cells in the fused droplet.SELECTED DRAWING: Figure 2

Description

Technical Field

[0001] Embodiments of the present invention relate to a method and system for producing target cells.

Background Art

[0002] In the field of regenerative medicine for producing cells by gene transfer, it is desired to produce highly uniform cells. For example, when producing iPS cells by introducing the Yamanaka factors, which are activators, into cells, it is necessary to introduce many types of genes into the source cells.

Summary of the Invention

Problems to be Solved by the Invention

[0003] The problem to be solved by the present invention is to provide a technique for improving the reaction efficiency, uniformly introducing an activator into source cells, and producing homogeneous target cells.

Means for Solving the Problems

[0004] The target cell production method according to the embodiment includes forming a first water-in-oil droplet containing source cells by mixing an aqueous solution containing source cells and an oil phase, forming a second water-in-oil droplet containing liposomes by mixing a liposome aqueous solution encapsulating an activator and an oil phase, fusing the formed first water-in-oil droplet and the second water-in-oil droplet, and introducing the activator into the source cells within the fused droplets.

Brief Description of the Drawings

[0005]

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Mode for Carrying Out the Invention

[0006] Hereinafter, embodiments will be described with reference to the accompanying drawings. In each embodiment, substantially the same constituent parts are denoted by the same reference numerals, and the description thereof may be partially omitted. The drawings are schematic, and the relationship between the thickness and the planar dimensions of each part, the ratio of the thicknesses of each part, etc. may be different from the actual ones.

[0007] (First Embodiment) The first embodiment is a method for producing target cells. The method includes forming a first water-in-oil droplet containing material cells by mixing an aqueous solution containing the material cells and an oil phase, forming a second water-in-oil droplet containing liposomes by mixing an aqueous liposome solution encapsulating an activator and an oil phase, fusing the formed first water-in-oil droplet and the second water-in-oil droplet, and introducing the activator into the material cells within the fused water droplets. This method includes introducing an activator into the material cells.

[0008] For example, as shown in FIG. 1, this method (a) Mix an aqueous liposome phase encapsulating an activator to be introduced into the material cells and an oil phase, for example, at a certain ratio and at a certain rate to continuously form first water-in-oil droplets containing liposomes (S11). (b) Mix an aqueous solution phase containing the material cells and an oil phase, for example, at a certain ratio and at a certain rate to continuously form second water-in-oil droplets containing the material cells (S12). (c) Sequentially fuse the continuously formed first water-in-oil droplets and the continuously formed second water-in-oil droplets to continuously obtain third water-in-oil droplets (S13), and (d) Introduce the activator into the material cells within each of the continuously obtained third water-in-oil droplets to continuously obtain target cells (S14). It includes.

[0009] The "target cells" are the cells to be produced. Examples of target cells can be any desired cells configured to have properties different from the original material cells by introducing an activator. Such cells may be, for example, any cells formed by introducing a gene. Examples of target cells can be cells used in medical fields such as laboratory level, genetic engineering, production of recombinant proteins, basic research fields, gene therapy, cell diagnosis, and regenerative medicine. In the case of cells used in regenerative medicine, for example, induced pluripotent stem cells (iPS cells) and somatic stem cells such as mesenchymal stem cells can be mentioned.

[0010] The "material cells" are the cells into which an activator is introduced to form target cells, and are selected according to the target cells to be obtained.

[0011] The "target cell" can be, for example, a cell in a state after contact between an activator and a source cell, a cell in a state after the action of an activator, a cell in a state after reaction with an activator, or a cell in a state after introduction of an activator. What kind of target cells to produce and / or recover may be determined by the implementer as desired. In some cases, the target cells may be used as further source cells, and in that case, it is possible to obtain further target cells by subsequently introducing an activator. In other words, for example, through a process in which a phenomenon occurs where, over time, an activator is applied to the source cells multiple times and ultimately becomes a target cell, target cells are formed from the source cells.

[0012] "Source cell" can be used interchangeably with the terms "initial source cell", "source cell at the first stage", and "seed cell". For example, when multiple activators are used and introduced repeatedly, the source cell is in the first stage before introduction, and by the time the final target cells are obtained, the properties of the source cell can change according to the number of repetitions. Here, for the sake of convenience, the term "stage" is used for its state, properties, or the presence or absence of contact with a specific activator, and it is also described as "source cell at the second stage", "source cell at the third stage"... "source cell at the nth stage" (n is an integer of 3 or more) according to the number of introductions of the activator. Whether a "source cell" that has made contact with a specific activator is considered a "source cell at the nth stage" or a "target cell" may be determined according to the desire of the implementer or the subsequent procedure. Depending on the selected source cells and / or target cells, differentiation is induced by the activator entering the system containing the source cells multiple times, and the desired target cells are formed. Also, depending on the selected source cells and / or target cells and as desired, the amount and timing of introducing the activator may be controlled.

[0013] Here, the "activator" may be any active ingredient having an effect of acting on and / or reacting with the material cells, thereby changing them into target cells having properties, physical properties, and / or forms different from those of the original material cells, transferring them to the target cells, or bringing them into the state of the target cells. Examples of the activator may be, for example, substances for recombining the genome of desired cells, and generally, substances referred to as genes. Specifically, for example, the activator may be a natural product, a compound, an extract, a nucleic acid, a peptide, a protein, etc. For example, the nucleic acid may be a nucleic acid fragment, a nucleic acid construct, DNA, RNA, etc. The activator applied to the material cells may be of one type or a combination of two or more types. For uniform introduction, it is preferable that the activator contained in one liposome be of one type. Examples of the case of using a combination of multiple types of activators will be further described in the embodiments described later.

[0014] A typical example of the activator is a nucleic acid, and for example, it may be a nucleic acid selected from the group consisting of a plasmid, an oligonucleotide, a polynucleotide, small interfering RNA (siRNA), microRNA (miRNA), DNA, an aptamer, and a ribozyme. Also, it may be an antisense oligonucleotide, an antagomir, aDNA, a plasmid, ribosomal RNA (rRNA), transfer RNA (tRNA), small nuclear RNA (snRNA), mRNA, etc., and different types of RNA and / or DNA may be used in combination.

[0015] A liposome can be a particle with a lipid bilayer membrane encapsulating an aqueous core. Any liposome known per se may be used for the liposome. For example, the lipid composition forming the liposome may contain the first lipid (FFT-10) of formula (I) and / or the second lipid (FFT-20) of formula (II) as its constituent components. Examples of using these lipid components will be further described in the embodiments described later.

Chemical formula

[0016] Examples of oils used as the oil phase can be, for example, fluorinated oils, mineral oils having a carbon chain, silicone oils, etc. Examples of fluorinated oils are, for example, fluorinated oils having fluorocarbons such as Fluorinert, etc. However, it is not limited thereto.

[0017] Examples of the aqueous solution can be, but are not limited to, buffer solutions, cell culture media, etc.

[0018] For example, examples of the combination of droplets (aqueous solution phase) and the outer liquid (oil phase) are not limited thereto, but are as follows: · A combination of an aqueous solution as droplets and a mineral oil having a carbon chain, a silicone oil, etc. as the outer liquid; · A combination of an aqueous solution as droplets and a fluorinated oil having fluorocarbons such as Fluorinert as the outer liquid.

[0019] In such a case, considering the combination of the oil and the aqueous solution, the presence or absence of addition of further components such as surfactants, and their types and amounts used may be selected. For example, when combined with a silicone oil, etc. or in the case of a combination with a fluorinated oil, it is also preferable to use a fluorinated surfactant in combination.

[0020] The formation of the first water-in-oil droplets can be carried out, for example, using a microchannel system 10 having three branches as shown in FIG. 2. The microchannel system 10 includes a microchannel having three branch portions. It also has a main trunk portion 11 and a branch portion 12. The branch portion 12 includes a first water-in-oil droplet formation channel 13 and a second water-in-oil droplet formation channel 14.

[0021] The first water-in-oil droplet formation flow path 13 includes an oil phase supply flow path 15 and an aqueous solution phase supply flow path 16. Here, the oil phase supply flow path 15 and the aqueous solution phase supply flow path 16 are connected at a right angle, but it is not limited to this. They may be connected so that both are in the same flow direction or so that both are in opposite flow directions. However, in order to generate droplets, an angle close to a right angle is preferable. The oil phase 17 is sent to the oil phase supply flow path 15 from the opening at its end. The cell-containing solution 18 is sent to the aqueous solution phase supply flow path 16 from the opening at its end. The cell-containing solution 18 is an aqueous solution containing the material cells 1 suspended therein. The aqueous solution containing the material cells 1 can be any medium, physiological saline, or physiological buffer solution according to the cells to be used. For example, by intermittently sending the cell-containing solution 18 into the oil phase 17 at a constant flow rate and / or a constant cell concentration, uniform water-in-oil droplets 19 having a uniform particle size containing the material cells uniformly or almost uniformly are continuously formed. In other words, by sending an aqueous solution containing cells at a constant concentration into the oil at a constant flow rate, the water-in-oil droplets 19 have a constant particle size, and the number of cells contained in the water-in-oil droplets 19 becomes constant.

[0022] The second water-in-oil droplet formation flow path 14 includes an oil phase supply flow path 20 and an aqueous solution phase supply flow path 21. Here, the oil phase supply flow path 20 and the aqueous solution phase supply flow path 21 are connected at a right angle, but it is not limited thereto. They may be connected so that both are in the same flow direction or so that both are in opposite flow directions. However, in order to generate droplets, an angle close to a right angle is preferable. The oil phase 17 is sent from the opening at the end of the oil phase supply flow path 20. The liposome-containing solution 22 is sent from the opening at the end of the aqueous solution phase supply flow path 21. Liposomes 2 are suspended and contained in the liposome-containing solution 22. The aqueous solution containing liposomes 2 can be any medium, physiological saline, or physiological buffer according to the cells to be used. For example, in the flow path, by intermittently sending out the liposome-containing liquid 22 into the oil phase 17 at a constant flow rate and / or a constant liposome concentration, the particle size of the water-in-oil droplets 24 can be made constant or substantially constant. Thereby, homogeneous water-in-oil droplets 24 containing liposomes 2 uniformly or substantially uniformly are continuously formed. That is, by making the particle size of the water-in-oil droplets 24 constant or substantially constant, the number of liposomes contained in the water-in-oil droplets 24 becomes constant. For example, the delivery of the aqueous solution phase 22 to the oil phase 17 may be such that the aqueous solution phase 22 and the oil phase 17 merge at the same speed and are sent downstream.

[0023] The water-in-oil droplets 19 containing the material cells 1 intermittently and continuously formed in the first water-in-oil droplet forming flow path 13 and the water-in-oil droplets 24 containing the liposomes 2 intermittently and continuously formed in the second water-in-oil droplet forming flow path 14 are respectively sent toward the oil phase 17 of the branch portion 12 and the main trunk portion 11, and reach the main trunk portion 11 at the same or substantially the same timing. Upstream of the main trunk portion 11, the two merge, and the water-in-oil droplets are fused with each other as numbers, for example, at a ratio of 1:1 to form one water droplet. For example, the fusion of the first water-in-oil droplet 19 and the second water-in-oil droplet 24 may be caused by changes in the conditions in the microchannel, such as the confluence of the microchannels, the change in pressure in the microchannel, the flow in the microchannel, such as the change in the liquid flow, the agitation of the liquid in the microchannel, etc. For example, the fusion may be performed at a certain ratio between the water droplets, for example, 1:1, and / or at a certain speed, a certain interval, a certain flow rate, and / or a certain amount. The fusion of the droplets may be promoted by a water droplet fusion mechanism or device that can cause an electric stimulus by an electrode, a physical stimulus by a change in the shape of the inner wall surface or the inner space of the flow path, a thermal stimulus by a temperature regulator, a magnetic stimulus such as a magnetic field by a magnetic force, etc., or the fusion may be performed by them. Alternatively, the water droplet fusion may be controlled by the type and concentration of the surfactant contained in the aqueous solution of the cell-containing solution 18 and / or the liposome-containing solution 22.

[0024] Subsequent to the water droplet fusion, an activator (not shown) contained in the liposome 2 within the water droplet in oil is introduced into and / or made to act on the material cell 1. In FIG. 2, an example is shown in which the activator contained in the liposome 2 is a gene. The “gene introduction part” in FIG. 2 is an example of the “activator introduction part”. It can be appropriately rephrased according to the type of activator. The introduction of the activator into the material cell 1 may be promoted by the above-described water droplet fusion mechanism or apparatus. Alternatively, separately from the water droplet fusion mechanism or apparatus, electrical stimulation by electrodes, physical stimulation by a change in the shape of the inner wall surface or inner space of the flow path, thermal stimulation by a heater or the like, magnetic stimulation such as a magnetic field by magnetism or magnetic force, light such as visible light, infrared light, and / or ultraviolet light, sound of a specific or arbitrary frequency, a sound wave such as ultrasonic waves, an activator introduction promotion mechanism or apparatus that can generate the like, for example, a gene introduction mechanism or apparatus, an activator action promotion mechanism or apparatus, etc. may be used to promote it. Further, the introduction of the activator may be stimulated or promoted, for example, by temperature control under certain conditions. For example, the introduction of the activator and / or the promotion of the action of the activator may be performed by adjusting the introduction, action, and / or reaction environment of the activator. Such adjustment of the introduction, action, and / or reaction environment of the activator can be performed, for example, by changing or maintaining at least one selected environmental condition from time, temperature, and gas concentration (for example, CO2 concentration, etc.). For example, the activator may be a substance that acts on the material cell and / or reacts with the material cell at least at any one of the time points before the introduction into the material cell, during contact with the material cell, at the time of introduction into the material cell, and after the introduction into the material cell. The introduction of the activator into the cell includes bringing the activator to a distance at which the activator can act on the cell, bringing the activator into the cell, bringing the activator into a state where it can act on the nucleus of the cell, bringing the activator into the nucleus of the cell, etc. Also, it may be either that the activator acts on the cell, that the activator reacts with the cell, or both. The introduction of the activator into the cell may be performed by incubation at a certain temperature. The incubation may be performed while being sent through the flow path, or may be performed while being held at a specific position by, for example, a cell capture mechanism or apparatus. For example, the incubation temperature may be about 35°C to about 39°C, etc., depending on the type of cell and activator.An activator is introduced, and the material cell 1 becomes the target cell 3. Finally, the target cells are collected.

[0025] The moving direction of the liquid in the microchannel 10 is indicated by an arrow in FIG. 2. The liquid moves from the end on the branch part 12 side toward the end on the main trunk part 11 side. The movement of the liquid may be caused by the pressure applied from the end on the branch part 12 side, or may be caused by the suction from the end on the main trunk part 11 side.

[0026] Such a method for producing target cells, in other words, for example, (1) a step of producing water-in-oil droplets containing cells; (2) a step of producing water-in-oil droplets containing liposomes encapsulating an activator; (3) a step of fusing two water-in-oil droplets; (4) a step of reacting the activator contained in the liposome with the cells in the fused water-in-oil droplets is a method of reacting liposomes and cells containing the above steps in a microspace. For example, it is also possible to produce target cells by sequentially introducing a plurality of activators into the material cells. In that case, for example, (1) to (4) may be repeated the number of times corresponding to the number of activators. In that case, as a result of the action or introduction of the activator for the first time, the material cell becomes a target cell, but this obtained target cell may be used as a second material cell that is different in state, property, etc., that is, at a different stage, from the initial material cell.

[0027] Also, for example, here, "sequentially introducing a plurality of activators into the material cells" may mean repeatedly introducing the same type of activator a plurality of times (two or more times) into the material cells sequentially. That is, in this case, (1) to (4) may be repeated the number of times the activator is introduced. Further, repeatedly introducing one type of activator a plurality of times and sequentially introducing a plurality of different types of activators may be combined as desired.

[0028] Summarizing the embodiments, for example, by this method, in a microchannel having a bifurcation where each one end is further bifurcated, an oil phase that flows with a certain amount of oil is formed from one end of one branch, and with respect to this oil phase, an aqueous solution containing material cells is intermittently poured in from one end of the other branch at a certain amount and a certain speed, whereby a plurality of first water-in-oil droplets containing material cells can be continuously formed in the oil phase. On the other hand, in the other branch, an oil phase that flows with a certain amount of oil is formed from one end thereof, and with respect to this oil phase, an aqueous solution containing liposomes is intermittently poured in from one end of the other branch at a certain amount and a certain speed, whereby a plurality of second water-in-oil droplets containing liposomes can be continuously formed in the oil phase. These are merged at a well-timed manner, and the water droplets are sequentially fused, and an activator contained in the liposomes is introduced. In this way, by encapsulating a certain number of cells and a certain number of liposomes in a uniform microvolume, it is possible to simultaneously realize control of the contact probability and suppression of the diffusion of liposomes, and it is possible to improve the gene introduction rate into cells. In addition, the cell introduction rate of the activator becomes uniform, and quality control becomes easy.

[0029] In the case of culturing in a conventional Petri dish, since it is difficult to uniformly introduce liposomes into each cell, a problem is that variations occur in the states of the obtained target cells. However, by reacting droplets containing liposomes in a certain volume with a certain number of, for example, one or more cells, it is possible to produce target cells in a uniform state.

[0030] (Second Embodiment) As a second embodiment, the liposomes used in the first embodiment will be described. Liposomes are particles of a lipid bilayer membrane that encapsulates an aqueous solution core, that is, lipid particles. Any liposomes known per se may be used for the liposomes. For example, the lipid composition constituting the lipid bilayer membrane forming the liposomes may contain, as its constituent components, a first lipid (FFT-10) of formula (I) and / or a second lipid (FFT-20) of formula (II).

Chemical formula

[0031] The lipid bilayer membrane, i.e., lipid particles, may contain additional lipids in addition to the above-described first lipid and second lipid. In the composition of the lipid molecular material constituting the lipid particles, the fraction consisting of the first lipid and the second lipid is hereinafter referred to as the "first fraction". Also, the fraction consisting of lipid molecular materials other than the first lipid and the second lipid is hereinafter referred to as the "second fraction". The lipids contained in the second fraction are collectively hereinafter also referred to as "third lipid".

[0032] The terms "first fraction" and "second fraction" represent the composition of the components of the lipid particles and do not indicate the physical positions of the lipids contained therein. For example, the components of the first fraction and the second fraction do not necessarily form one aggregate each in the lipid particles, and the lipids contained in the first fraction and the lipids contained in the second fraction may be mixed and present. The blending ratio of the first fraction with respect to the entire lipid material constituting the lipid particles can be 5% or more, 10% or more, 15% or more, for example, 10% to 80%, or 15% to 60%, etc.

[0033] In other words, the total content of FFT-10 and FT-20 can be, for example, 5% or more, 8% or more, 10% or more, 15% or more, for example, 10% to 80%, or 15% to 60%, 15% to 50%, etc., as the blending ratio of the lipid particles. The maximum content of FFT-10 and FFT-20 in the lipid particles may be, for example, an amount such that the lipid particles can form liposomes. The blending ratio of the second lipid 1b in the first fraction may be 0% or more to 100%, and can be, for example, 15% to 75%, 20% to 60%, 24% to 50%, etc. Similarly, the blending ratio of the first lipid 1a in the first fraction may be 0% or more to 100%, and can be, for example, 15% to 75%, 20% to 60%, 24% to 50%, etc.

[0034] Depending on the mixing ratio of the first lipid and the second lipid in the first fraction, the particle size and permeability of the lipid particles into cells may change. For example, the larger the amount of the second lipid, the larger the particle size of the lipid particles can be. The average particle size of the lipid particles can be changed according to the application. For example, it may be adjusted to about 50 nm to about 300 nm. For example, it may be about 70 nm to about 100 nm.

[0035] The type of the third lipid contained in the second fraction of the lipid particles is not limited. For example, the second fraction contains a base lipid. As the base lipid, for example, a lipid that is a main component of a biological membrane can be used. The base lipid can be a phospholipid or a sphingolipid, such as dipalmitoylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, kephalin, or cerebroside, or a combination thereof.

[0036] For example, as the base lipid, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-stearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC), 1,2-di-O-octadecyl-3-trimethylammonium propane (DOTMA), 1,2-dioleoyl-3-dimethylammonium propane (DODAP), 1,2-dimyristoyl-3-dimethylammonium propane (14:0 DAP), 1,2-dipalmitoyl-3-dimethylammonium propane (16:0 DAP), 1,2-distearoyl-3-dimethylammonium propane (18:0 DAP), N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propane (DOBAQ), 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), or cholesterol, or a combination thereof, etc. is preferably used.

[0037] As the base lipid, it is particularly preferable to use a cationic lipid or a neutral lipid, and the acid dissociation constant of the lipid particles can be adjusted by its content. It is preferable to use DOTAP as the cationic lipid and DOPE as the neutral lipid.

[0038] The second fraction preferably also contains a lipid that prevents aggregation of the lipid particles. For example, the lipid that prevents aggregation may further contain a PEG-modified lipid, such as polyethylene glycol (PEG) dimyristoyl glycerol (DMG-PEG), a polyamide oligomer derived from an omega-amino (oligoethylene glycol) alkanoic acid monomer (U.S. Patent No. 6,320,017), or monosialoganglioside, etc.

[0039] The second fraction may further contain a lipid having relatively low toxicity for adjusting toxicity; a lipid having a functional group for binding a ligand to the lipid particles; a lipid for suppressing leakage of inclusions such as a sterol, such as cholesterol. In particular, it is preferable to include cholesterol.

[0040] The type and composition of the lipid used in the second fraction may be appropriately selected in consideration of the acid dissociation constant (pKa) of the target lipid particles or the particle size of the lipid particles, the type of the included activator, or the stability in cells, etc.

[0041] For example, when the second fraction contains DOPE, DOTAP, cholesterol, and DMG-PEG, it is preferable because the delivery efficiency of the active agent is particularly excellent.

[0042] In addition to the active agent, further components may be encapsulated in the lipid particles as needed. The further components are, for example, pH adjusters, osmotic pressure adjusters, gene activators, and the like. The pH adjuster is, for example, an organic acid such as citric acid and its salts. The osmotic pressure adjuster is sugar, an amino acid, or the like. Here, the gene activator can be any substance that promotes or supports the activity of the active agent when the active agent is a gene.

[0043] The lipid particles encapsulating the active agent and other substances as needed can be produced, for example, by known methods used when encapsulating small molecules in lipid particles, such as the Bangham method, the organic solvent extraction method, the surfactant removal method, or the freeze-thaw method. For example, a lipid mixture obtained by including the materials of the lipid particles in an organic solvent such as alcohol at a desired ratio and an aqueous buffer containing components to be encapsulated such as an activator are prepared, and the aqueous buffer is added to the lipid mixture. By stirring and suspending the obtained mixture, lipid particles encapsulating the activator and the like are formed. The lipid particles thus obtained are an example of liposomes.

[0044] By using such liposomes, it is possible to improve the gene transfection rate into cells. In addition, the cell transfection rate of the active agent becomes more uniform, and quality control becomes easier.

[0045] (Third Embodiment) The third embodiment is a further example of the microchannel system shown in the above-described first embodiment. An example of the microchannel system will be described with reference to FIG. 3. The microchannel system 30 includes a first water droplet generation unit 31, a second water droplet generation unit 32, a confluence unit 33, a water droplet fusion unit 34, and an activator introduction unit (gene introduction unit) 35. In the first water droplet generation unit 31, a water-in-oil droplet 19 containing the material cells 1 is formed. In the second water droplet generation unit 32, a water-in-oil droplet 24 containing the liposomes 2 is formed. The confluence unit 33 is connected to the first water droplet generation unit 31 and the second water droplet generation unit 32, and the oil phase containing the water droplet 19 containing the material cells 1 and the oil phase containing the water droplet 24 containing the liposomes 2 are made to converge. In the water droplet fusion unit 34 connected to the confluence unit 33, the water droplet 19 containing the material cells 1 and the water droplet 24 containing the liposomes 2 are fused within the oil phase. The activator introduction unit (gene introduction unit) 35 is connected to the water droplet fusion unit 34, receives the fused water droplets from the water droplet fusion unit 34, and, for example, incubates them. Thereby, an activator (for example, a gene) is introduced into the cells 1 to produce the target cells 3.

[0046] The microchannel system 30 produces target cells by introducing an activator into the material cells. The microchannel system 30 includes minute channels. The first water droplet generation unit 31 includes a first water-in-oil droplet formation channel 13, a first oil phase supply channel 15, and a first aqueous solution phase supply channel 16 that converges therewith. Oil 17 serving as an oil phase is sent to the first oil supply channel 15 from the opening at its end. An aqueous solution 18 serving as an aqueous solution phase is sent to the first aqueous solution phase supply channel 16 from the opening at its end. The aqueous solution 18 is a material cell suspension and contains the material cells.

[0047] The second water droplet generation unit 32 includes a second water-in-oil droplet formation channel 14, a second oil phase supply channel 15, and a second aqueous solution phase supply channel 21 that converges therewith. An aqueous solution 22 supplied from the opening at its end to the second aqueous solution phase supply channel 21 is a liposome-containing solution and contains liposomes encapsulating an activator.

[0048] In the confluence section 33, the first oil-in-water droplet formation flow path and the second oil-in-water droplet formation flow path merge and are connected, and are configured to send the oil-in-water droplets 19 from the first oil-in-water droplet formation flow path and the oil-in-water droplets 24 from the second oil-in-water droplet formation flow path to the water droplet fusion section 34. The water droplet fusion section 34 fuses the first oil-in-water droplet 19 and the second oil-in-water droplet 24. The fused water droplet 25 generated by the fusion contains both the liposome 2 and the material cell 1.

[0049] The water droplet fusion section 34 includes a flow path following the activator introduction section 35. In FIG. 3, the activator introduction section 35 is shown as a gene introduction section 35 as an example. In the activator introduction section 35, the liposome 2 encapsulating the activator and the material cell 1 are brought into contact to introduce the activator into the material cell 1. The activator introduction section 35 may be provided with a temperature control mechanism, or a temperature adjustment mechanism or device for heating or cooling the entire flow path of the microchannel system or partially as desired may be arranged. The activator induction section 35 may be provided with a condition adjustment mechanism or device for maintaining specific conditions until the cells contained therein change into the desired target cells 3. The obtained target cells 3 can be recovered from the opening at the end of the flow path. Such a microchannel system can be configured as a flow path formed on a substrate such as glass, resin, or silicon. Alternatively, it may be composed of a hard or soft tube or the like. Further, the microchannel system 30 may further include a mechanism or device for promoting the recovery of target cells. Such a recovery promotion mechanism / device may be a cell capture mechanism / device or a mechanism or device for promoting the removal of oil from the oil phase and moisture from the water droplets. For example, physical capture means such as filters, steps, obstacle structures, slits, and membranes may be arranged around the opening.

[0050] The water droplet fusion part 34 may be provided with a water droplet fusion mechanism or device (not shown) that promotes the fusion of water droplets. Examples of the water droplet fusion mechanism or device can be a mechanism or device that can generate electrical stimulation by an electrode, physical stimulation by a change in the shape of the inner wall surface or inner space of the flow path, thermal stimulation by a heater, etc., and magnetic stimulation such as a magnetic field by magnetism or magnetic force. The water droplet fusion mechanism or device (not shown) may be provided with an introduction mechanism or device (not shown) that stimulates or promotes the introduction of the activator. The water droplet fusion mechanism or device and the introduction mechanism or device may share one mechanism or device. Also, if desired, part or all of the microchannel system may be temperature-controlled by a temperature control mechanism or device such as a heater or a temperature regulator.

[0051] By using the microchannel system, it is also possible to easily control the flow rate and / or flow velocity by flowing the liquid in the flow path at a constant speed or applying a constant pressure. However, the microchannel system 30 may further include a control unit 35 that controls the movement and operation of each part of the microchannel system. For example, the control unit 35 may include a movement mechanism (not shown) involved in the movement of oil or aqueous solution in the flow path, and a timing adjustment mechanism (not shown) that adjusts the movement of the operations and processes performed in each part of the microchannel system 30. Also, a sensor (not shown) for observing or detecting the operations and processes in the microchannel system 30 may be provided.

[0052] (Fourth Embodiment) The fourth embodiment is an example of a method for producing target cells for sequentially introducing two types of active ingredients. When sequentially introducing two types of active ingredients into one material cell, for example, the method of the first embodiment may be repeated twice. In that case, the material cell into which the first type of active ingredient has been introduced is called the material cell in the second stage, assuming it is a cell different from the initial material cell. On the other hand, the initial material cell is also referred to as the material cell in the first stage. For example, when using the microchannel system 30 according to the third embodiment, the cells obtained as the target cells 3 by the first introduction are collected, and by using them as the second material cells and further introducing the second active ingredient, it is possible to finally obtain the desired target cells 3.

[0053] As shown in FIG. 4, an example of a method for producing target cells according to the fourth embodiment includes sequentially introducing first and second activators into a material cell. For example, the method for producing target cells may include the following: (a’) Mixing a first liposome aqueous solution phase containing a first activator to be introduced into the material cell in the first stage with an oil phase at a certain ratio and at a certain rate, and continuously forming first water-in-oil droplets containing liposomes (S41); (b’) Mixing an aqueous solution phase containing the material cell with an oil phase at a certain ratio and at a certain rate, and continuously forming second water-in-oil droplets containing the material cell (S42); (c’) Sequentially fusing the continuously formed first water-in-oil droplets and the continuously formed second water-in-oil droplets to continuously obtain third water-in-oil droplets (S43); (d’) Inside the continuously obtained third water-in-oil droplets, the activator is sequentially introduced into the material cell to continuously obtain the material cell in the second stage (S44); (e) Mixing a second liposome aqueous solution phase containing a second activator to be introduced into the material cell with an oil phase at a certain ratio and at a certain rate, and continuously forming fourth water-in-oil droplets containing liposomes (S45); (f) Sequentially fusing the third water-in-oil droplets containing the material cell in the second stage obtained continuously and the continuously formed fourth water-in-oil droplets to continuously obtain fifth water-in-oil droplets (S46); and (g) Inside the continuously obtained fifth water-in-oil droplets, the second activator is sequentially introduced into the material cell in the second stage to continuously obtain target cells (S47).

[0054] Such a method is a method for continuously producing a plurality of target cells, whereby it is possible to produce a plurality of cells into which an activator has been uniformly introduced. Also, such a method can efficiently and / or homogeneously produce target cells.

[0055] (Fifth Embodiment) An example of a microchannel system 50 for carrying out the fourth embodiment will be described with reference to FIG. 5. The microchannel system 50 is a system for producing target cells by sequentially introducing two types of activators into the material cells, that is, in two steps. Basically, it has the configuration of the microchannel system 30 which is the third embodiment, and further includes a configuration for introducing the second activator into the cells. Such a configuration includes a third water droplet generation unit 32b for forming a water-in-oil droplet 24b containing liposomes 2b containing the second active ingredient, a second-stage water droplet fusion unit 34b, an activator introduction unit (gene introduction unit) 35b, and the like. In FIG. 5, for the configuration for the first activator, the letter "a" is appended to the end of each reference numeral. Since the configuration for the second activator is basically the same as the configuration for the first activator, the letter "b" is appended to the end of each reference numeral. In FIG. 5, as an example of the stimulation mechanism 53, an example is shown in which an arbitrary water droplet fusion device is arranged in the water droplet fusion parts 34a and 34b so as to sandwich the flow path. For example, the stimulation mechanism 53 may be arranged in the gene introduction parts 35a and 35b, or may be arranged in the water droplet fusion parts 34a and 34b and the gene introduction parts 35a and 35b. Such a microchannel system can be configured as a flow path formed on a substrate such as glass, resin, or silicon. Alternatively, it may be configured with a hard or soft tube or the like. Further, in such a microchannel system, a main flow path 52 where contact between cells and liposomes and introduction of an activator are mainly performed may exist like a backbone supporting the system.

[0056] According to the fifth embodiment, it is possible to continuously perform the introduction of two types of activators, that is, the introduction of the first activator into the material cells at the first stage and the introduction of the second activator into the material cells at the second stage obtained thereby. Such a system can continuously produce a plurality of target cells. Thereby, it is possible to produce a plurality of cells into which the activator has been introduced homogeneously. Also, such a system makes it possible to manufacture target cells more efficiently and / or with higher quality. For example, by mounting a control unit and a storage unit such as a computer or a CPU, as well as a pre-stored program, table, timing mechanism or device, and a timing observation mechanism or device, it is possible to manufacture target cells with high throughput, for example, fully automatically.

[0057] By performing the reaction process between the liposome encapsulating the gene and the cell inside the water-in-oil droplet, diffusion can be suppressed, the reaction efficiency can be improved, and homogeneous cells can be manufactured. For example, it becomes possible to uniformly introduce a plurality of genes, the reaction of bringing the liposome into contact with the cell is made uniform, the separability of the step of bringing two different liposomes into contact is enhanced, and for example, it also becomes possible to manufacture iPS cells homogeneously.

[0058] (Sixth Embodiment) The sixth embodiment shows an example of a microchannel system that sequentially introduces n types (where n is an integer of 3 or more) of activators into material cells in n steps to produce target cells. It is possible to finally obtain target cells while forming material cells at the (n - 1)-th stage as intermediates every time each activator is introduced. Such a microchannel system, in the microchannel system 50 shown in FIG. 5, for all of the n types of activators used, in order to form water-in-oil droplets respectively, it only needs to be provided with the required number of additional water droplet generation parts for n types. The basic structure of the microchannel system 50 is, for example, a flow channel formed on a substrate. Looking at the whole from above, one oil-phase flow channel becomes a main trunk flow channel 52 like a backbone, and its most upstream side branches into two. One of the branches extends to a water-in-oil droplet formation flow channel 13 for material cell 1, and the other branch extends to a water-in-oil droplet formation flow channel 14a for the first activator. Downstream of the main trunk flow channel 52, n - 1 oil-phase flow channels 20b to 20n - 1 (not shown for those other than 20b) merge respectively. Aqueous solution phase flow channels 21b to 21n (not shown for those other than 21b) merge into those oil-phase flow channels respectively at their upstream. In other words, water-in-oil droplet formation flow channels 14b for the second activator to water-in-oil droplet formation flow channels 14n for the n-th activator (not shown) merge into the main trunk flow channel 52 through their respective oil-phase flow channels. Water-in-oil droplets formed in their respective water-in-oil droplet formation flow channels are sent to the main trunk flow channel 52. The material cell 1 contained in the first water-in-oil droplet 19 formed at the most upstream rides on the flow of the oil phase in the main trunk flow channel 52 and is sent downstream while being sequentially stimulated by the first activator to the n-th activator. As a result, the stage of the material cell changes sequentially from the initial stage (the first stage) to the second stage, the third stage, and so on. For example, when the activator is a gene, n types of genes are sequentially introduced into the material cell and, in some cases, transformed. The material cell 1 is introduced with activators step by step in this way, and finally, the target cell 3 is obtained.

[0059] One example of such a microchannel system may have the following configuration. The microchannel system is for introducing the first to the nth activators into the material cells (where n is an integer of 3 or more) to produce target cells. The microchannel system includes a first water-in-oil droplet formation channel, a second water-in-oil droplet formation channel, a first fusion part, a first activator introduction part, the third to the n + 1th water-in-oil droplet formation channels, the second to the nth fusion parts, and the second to the nth activator introduction parts. Such a microchannel system may include the minimum repeating unit "(cell supply 1) → water-in-oil droplet formation channel for liposomes containing an activator → fusion part → activator introduction part → (cell supply 2)" repeated the desired number of times. At each fusion part, the water-in-oil droplets 24a to 24n each containing liposomes 2a to 2n and the water-in-oil droplets 24a to 24n each containing material cells 1a to 1n are respectively fused to form fused droplets 25a to 25n. In each of the fused droplets 25a to 25n, the activators a to n are introduced into the material cells 1a to 1n, and the material cells 1b to 1n in the next stage are formed, or the target cells 3 are formed.

[0060] Such a microchannel system has, for example, the following configuration; · The first water-in-oil droplet formation channel prepares a first water-in-oil droplet containing a cell suspension containing the material cells in the first stage in an aqueous phase. The first water-in-oil droplet formation channel includes a first oil phase supply channel and a first aqueous phase supply channel that merges therewith; · In the second water-in-oil droplet formation channel, a second water-in-oil droplet containing a first liposome encapsulating a first activator is formed in an aqueous phase. The second water-in-oil droplet formation channel includes a second oil phase supply channel and a second aqueous phase supply channel that merges therewith. The second oil phase supply channel merges with the first oil phase supply channel on its downstream side. From the confluence point, an oil phase channel further extends downstream as a backbone structure or a main trunk channel; · At the first fusion part, the first water-in-oil droplet and the second water-in-oil droplet fuse with each other to become a first fused droplet. The first fusion part is located in a region extending downstream from the confluence point of the first water-in-oil droplet formation channel and the second water-in-oil droplet formation channel. For example, one example of the first fusion part is located on the downstream side of the confluence part; · In the first activator introduction section, the first liposome contacts the material cell, and the activator encapsulated in the first liposome is introduced into the material cell. Thereby, the material cell at the second stage is generated. The first activator introduction section is located downstream of the first fusion section; · Downstream of the first activator introduction section, a third oil phase supply path for introducing the second activator merges. Thereafter, downstream, the oil phase supply channels for introducing all the activators up to the nth activator respectively merge into the backbone oil phase channel; · Each of the third to (n + 1)th water-in-oil droplet formation channels includes a third to (n + 1)th oil phase supply channel and a third to (n + 1)th aqueous solution phase supply channel that merges therewith respectively. Through each of the third to (n + 1)th oil phase supply channels, each of the third to (n + 1)th water-in-oil droplet formation channels is connected and located downstream of the first to (n - 2)th activator introduction sections in sequence. The downstream sides of the third to (n + 1)th oil phase supply channels respectively merge into the main flow channel. In each of the third to (n + 1)th water-in-oil droplet formation channels, third to (n + 1)th water-in-oil droplets are formed. Each of the third to (n + 1)th water-in-oil droplets contains the second to nth liposomes. The second to nth liposomes respectively encapsulate the second to nth activators; · Each of the second to nth fusion sections is arranged downstream of the first to (n - 1)th activator introduction sections. In the second to nth fusion sections, the oil droplets containing the material cells at the second to (n - 1)th stages from the first to (n - 1)th activator introduction sections and the third to (n + 1)th water-in-oil droplets containing the second to nth activator-containing liposomes from the third to (n + 1)th water-in-oil droplet formation channels are respectively fused; · In the second to nth activator introduction sections connected to the second to nth fusion sections, the second to nth liposomes and the material cells at the second to (n - 1)th stages contained in the respective fused water droplets are respectively brought into contact, and the respective activators are introduced into the material cells at the corresponding stages. Thereby, the material cells or target cells at the third to nth stages are formed. · The formed material cells are maintained in water-in-oil droplets and sent to the next introduction. The formed target cells are recovered. The recovery of the target cells may be, for example, from an opening provided at the most downstream of the main flow path, or may be sent to a recovery container connected to the downstream of the main flow path and recovered. For the case where the size of the droplets moving in the system increases due to the fusion of the water-in-oil droplets, the microchannel system may include a recovery mechanism or a liquid removal mechanism for recovering the material cells at one end or removing the liquid from the water droplets to miniaturize. Such a mechanism may be, but is not limited to, means using shapes such as filters, sieves, nets, grilles, unevenness, or means using centrifugal force or suction force, means using electrodes or magnets, etc.

[0061] According to such an embodiment, it is possible to improve the reaction efficiency, uniformly introduce the activator into the material cells, and produce homogeneous target cells.

[0062] (Seventh Embodiment) The seventh embodiment is a microchannel system that provides a shape difference in the flow path structure between the fusion part and its upstream and downstream as a fusion mechanism in the fusion part. It can be the same as the microchannel system of other embodiments except for having a fusion structure with a shape difference in the fusion part.

[0063] An example of a fusion structure will be described with reference to FIG. 6. FIG. 6 shows a schematic view when the fusion part and the flow path parts before and after it (including somewhat the upstream and downstream regions thereof) are cut out for convenience and viewed from above. FIG. 6(a) is an example in which a region having a width (w2) wider than the width (w1) of the flow path of other parts is provided in the fusion part. In FIG. 6(a), the width of w2 is an example where it is five times the length of the width of w1. It is an example in which two trapezoidal bases are directed inward and the inside of the flow path is extended outward from the ends of the flow paths before and after. By temporarily widening the width, it is possible to approach subsequent droplets and promote fusion. FIG. 6(b) is an example in which two extensions of FIG. (a) are arranged in series in the flow direction of the flow path, that is, in the flow direction of the flow path. It is also called an hourglass type. In this case, while approaching subsequent droplets, on the one hand, after the flow path becomes wide, pressure can be generated by narrowing it primarily. Thereby, fusion is promoted. FIG. 6(c) is an example in which extension parts are formed as two triangles (rhombuses) with their bases facing each other in a direction perpendicular to the flow from the ends of the flow paths before and after, and FIG. 6(d) is an example in which the rhombuses are arranged in series in the flow direction. These can obtain the same effects as FIGS. 6(a) and (b) respectively. FIGS. 6(e) and (f) promote fusion by applying pressure to the water droplets moving the inner diameter of the flow path by folding the flow path located in the fusion part twice at 45° so as to be Z-shaped. Also, by attaching such an R to the flow path, intrusion of bubbles can be prevented. Also, FIGS. 6(e) and (f) are examples in which a space is created between the flow paths that bend and face each other at 45°, and the space is made into a flow path shallower than other parts. By forming a space, a symmetrical structure can be formed such that the flow is in a straight line. Also, as in FIG. 6(b), a part of the flow path may be made shallow (FIGS. 6(g), (h)). The shallow part is indicated by hatching. The depth d2 of that part is shallower than the normal depth d1 of the flow path. By narrowing a part of the width of the flow path part with the normal depth d1 to w3 above that, a so-called hourglass structure is formed. The depth d2 of the region indicated by hatching is, for example, about one-third of the depth d1 of other flow paths, which is effective, but is not limited thereto. By providing a shallow part in part, it becomes difficult for bubbles to enter. Also, by making it shallow while narrowing the width, fusion can be promoted more. For example, even by simply providing a step in the depth direction, it is possible to induce fusion.

[0064] (Eighth Embodiment) The eighth embodiment is a further example of the flow path design corresponding to the activator introduction part as shown in FIG. 7. In the activator introduction part, a desired activator contained in the water-in-oil droplets is introduced into the material cells by inclusion liposomes. For example, in such an activator introduction part, the introduction of the activator may be achieved by incubating the fused water droplets formed at the fusion part under arbitrary conditions. For example, an example of the activator introduction part provided with such an incubation mechanism is shown in FIG. 7. Other than such a configuration, it may be the same as any of the above-described embodiments.

[0065] This will be described with reference to FIG. 7. FIG. 7 shows a schematic view seen from above in a form in which the activator introduction part and the flow path parts before and after it (including somewhat the upstream and downstream regions thereof) are cut out for convenience. The flow path of the activator introduction part can be, for example, a linear shape along the flow direction of the flow path as shown in FIG. 2 or the like. However, by increasing the flow path volume, the incubation time of the cells may be controlled. For example, a meandering structure or a structure with a wider width may be provided. For example, as shown in FIG. 7(a), it may be designed to fold back a desired number of times in a direction perpendicular to the flow direction of the flow path. Also, for example, as shown in FIG. 7(b), as in FIG. 6(a), the width of a part of the flow path may be made wider compared to the width w1 of the flow path in other regions (w2).

[0066] As a further example of the incubation mechanism, a structure for capturing droplets may be provided. Examples of the capture structure are shown in FIGS. 7(c) and (d). The incubation mechanism of FIG. 7(c) is an example with a wider width as in FIG. (b). Specifically, this example is a flow path having regions where the flow path space extends in a trapezoidal shape from the side surfaces of the flow path toward both sides in a direction perpendicular to the flow direction, and a plurality of cell capture structures are arranged on the bottom surface of the flow path (FIG. 7(c-1)). The cell capture structure can be a protrusion that can stably capture cells. The example of FIG. 7(c-1) combines two protrusion parts with mirror-symmetrically contrasting structures as one capture unit. In this example, two protrusions are set as one set, and nine sets are arranged in the flow direction and three sets are arranged in the direction perpendicular to the flow. FIG. 7(c-2) is a perspective view showing an enlarged view of one set of the cell capture structures shown in FIG. 7(c-1). One protrusion is a rectangular parallelepiped that is long in the depth direction with a part missing in the depth direction. When two are arranged as a capture unit, the missing part becomes a concave portion like a part where a cylinder is bisected in the axial direction. Cells are stably captured at that part. However, the specific shape is not limited to this. Also, for example, the cell capture structure is not limited to the bottom surface (or lower surface) of the flow path, and may be arranged on the ceiling surface (upper surface) or both sides thereof. For example, an appropriate arrangement can be selected according to the combination of the droplet and the solvent. For example, when using something with a specific gravity lighter than the aqueous solution like mineral oil, it is desirable to install the capture structure on the lower surface, and when using something with a specific gravity heavier than the aqueous solution like fluorinate, it is desirable to install the capture structure on the upper surface. The size of the capture structure may be changed according to the size of the droplets to be produced. For example, it may be designed so that the diameter of one capture unit is about 10 μm to about 1 mm. Also, for example, the incubation mechanism may be a recess formed on the bottom surface of the flow path. Such a recess may be a micro-well structure, and a plurality of micro-pieces forming micro-wells may be arranged. For example, the size of one micro-well can have a diameter of about 10 μm to about 1 mm. An example of the arrangement of the micro-wells can be the arrangement shown in the protrusion set described in FIG. 7(c-1), but is not limited thereto. According to the above embodiments, a stable reaction and an efficient reaction are possible.

[0067] (Embodiment 9) Embodiment 9 is a method of continuously introducing an activator into a material cell. The method includes: forming a first water-in-oil droplet containing a material cell by mixing an aqueous solution containing the material cell and an oil phase, forming a second water-in-oil droplet containing liposomes by mixing an aqueous liposome solution encapsulating the activator and an oil phase, fusing the formed first water-in-oil droplet and the second water-in-oil droplet, and introducing the activator into the material cell within the fused water droplet. Specific details are as described above. Also, for example, by repeating some or all of these steps as described above, it is possible to sequentially introduce a plurality of activators into one cell. Alternatively, it is possible to introduce the same type of activator multiple times. Furthermore, it is possible to change the type of liposome used for each introduction and for each activator. The formation of water-in-oil droplets can be carried out by using the flow of each phase into microflows and their confluence. For example, it is also preferable to use a swirl flow path at that time. Swirl refers to, for example, a swirling flow generated around the cylinder axis in a piston mechanism, but any microflow path that causes such a swirling flow to occur may be used. According to such an embodiment, it is possible to improve the reaction efficiency, uniformly introduce the activator into the material cell, and produce homogeneous target cells.

[0068] Experimental Example Example 1 Formation test of water-in-oil droplets by a microchannel system (1) Fabrication of the microchannel system Using a microchannel system, water droplets in oil were formed. First, a microchannel system 80 as shown in FIG. 8 was constructed. FIG. 8(a) is a schematic plan view of the microchannel system 80, FIG. 8(b) is a cross-sectional view when the microchannel system 80 in FIG. (a) is cut along the line b-b', and FIG. 8(c) is a cross-sectional view when the microchannel system 80 in FIG. (a) is cut along the line c-c'. The microchannel system 80 is composed of two substrates stacked together. One of them is the substrate 81. The substrate 81 is a glass plate with a size of 5 cm × 3 cm × 3 mm. It has a groove 82 with a depth of 200 μm formed on one surface of one substrate 81. The substrate 81 has a further substrate with the same size as the substrate 81 on the groove side as the cover 83. The substrate 81 and the cover 83 are constructed by overlapping them so as to be adhered or fixed to each other. Openings 84 directed upward are provided at both ends of the groove. Depending on the shape of the groove 82, a flow path 85 is defined. The flow path 85 includes one bifurcated end, a hexagonal annular structure 86 arranged in series in the flow direction provided on the downstream side of the confluence point, a straight structure downstream thereof, and openings 84 at each end of the cover 83. Here, the hexagonal annular structure 86 is a swirl flow path. Syringe tubes 87 are respectively attached to the edges of the three openings 84 of the cover 83. The tube 87a extending from one of the upstream openings 84 reaches the inside of a container 88 that stores oil, and the tube 87b from the other upstream opening 84 reaches the inside of a container 89 that stores an aqueous solution. The tube 87c from the downstream opening 84 is connected to the tip of the cylinder of the syringe 81 via a joint 90. By the way, instead of the syringe, a liquid feeding mechanism or device such as a peristaltic pump may be used to achieve liquid feeding in the flow path.

[0069] As shown in FIG. 8(c), the flow path 85 includes seven partially shallow regions 92. Such regions may be made shallower by reducing the depth compared to the regions of other grooves when forming grooves in the substrate. Thereby, a shallow region 92 with a smaller depth, i.e., shallower than other regions, is obtained. By providing the shallow region 92, liquid mixing is well performed. One of such regions 92 is provided in the aqueous solution side flow path immediately before the branch on the aqueous solution side and the branch on the oil side merge. The other six regions 92 are provided in the annular structure 86. Oil from the container 88 and aqueous solution from the container 89 are mixed to form water-in-oil droplets. Incidentally, the annular region 86 can also be used as the water droplet fusion mechanism in the above-described embodiment. The shallow region 92 is provided at the depth of two non-adjacent sides of the hexagon constituting the annular structure 86.

[0070] (2) Liposome water-in-oil droplet generation test Rhodamine PE lipid (manufactured by Avanti) was added to the lipid composition of No. 43 at a molar ratio of 0.1% so that it could be visualized by fluorescence, and this was suspended in a HEPES solution to a concentration of 5%. This was regarded as a liposome-suspended aqueous solution and used as the aqueous solution side. As the oil side, FC40 (fluorinate, manufactured by 3M) was prepared. These were respectively accommodated in containers 88 and 89. The plunger of the syringe 81 was pulled, and each liquid was fed while applying a negative pressure inside the flow path. The inside of the flow path 85 was observed with an optical microscope from above in bright field and fluorescence field (wavelength 546 nm).

[0071] The results are shown in Fig. 9. The observation site is the region immediately upstream of the flow path and immediately after the confluence of the oil phase and the aqueous solution phase. The inner diameter of the flow path is 600 μm. Fig. 9(a) is a group of images taken over time in bright field, and Fig. 9(b) is a group of images taken over time in fluorescence field. The arrows in the figure indicate the direction of oil flow and the direction of aqueous solution flow, respectively. By pulling the plunger of syringe 91, it was observed that the oil phase from container 88 and the liposome-suspended aqueous solution from container 89 constantly moved downstream in flow path 85. At the same time, water droplets were generated as water-in-oil droplets 95a in the oil phase (Fig. 9(a-i), (b-i)), gradually swelled 95b (Fig. 9(a-ii), (b-ii)), grew 95c (Fig. 9(a-iii), (b-iii)), and finally became independent from the aqueous solution as the mother body and finally became one droplet (water droplet) 95d in the oil, and this state was clearly observed. From this result, it was shown that water-in-oil droplets can be formed using a microchannel structure.

[0072] (3) Homogeneity test of water-in-oil droplets Subsequently, water-in-oil droplets were continuously formed by the same method as in (2) above, and the fluorescence intensity of each droplet was observed. The results are shown in Fig. 10. Fig. 10(a) is an image taken under a microscope in a fluorescence field. By analyzing this image, for the fluorescence intensity of 15 water-in-oil droplets in the flow path, the fluorescence intensity of the cross section along line A-B in Fig. 10(a) and the moving average of the 20 pixels before and after were obtained. Fig. 10(b) plots the obtained fluorescence intensity on the vertical axis and the pixel position information corresponding to line A-B on the horizontal axis. Also, the data was corrected by parallelly moving the graph downward in the vertical axis direction so that the initial value became 0.

[0073] As a result, it became clear that the fluorescence amount (i.e., the liposome amount) of each droplet (i.e., water-in-oil droplet) was approximately equal. The fluorescence intensity at 80 - 120 pixels was 22.4 ± 1.34. From these facts, it became clear that the microchannel system can form water-in-oil droplets with uniform particle sizes and can evenly incorporate liposomes into a plurality of water-in-oil droplets.

[0074] Example 2: Simultaneous encapsulation of fluorescent beads and liposomes into water droplets in oil using a microchannel system (1) Fabrication of the microchannel system and simultaneous encapsulation test into water droplets in oil A test was conducted to confirm that two components, fluorescent beads and liposomes, can be simultaneously encapsulated into water droplets in oil using a microchannel system. Since the fluorescent beads are the same size as general cells, they were used as a substitute for cells.

[0075] The microchannel system used is shown in Fig. 11. The microchannel system 110 has an additional oil-phase channel connected up to the downstream region of the annular structure 86 (an example of a swirl channel) of the channel 85 in the microchannel system 80 (Fig. 8(a)) shown in Fig. 8. The channel 111 of the microchannel system 110 has, in its upstream part, a structure from a little downstream of the annular structure 86 from the channel 85 and an additional annular structure connected thereto. This additional annular structure is also a swirl channel in which seven shallow regions 92 are provided, similar to the annular structure 86. The flow of liquid into the channel 111 is as follows. First, in the region corresponding to the channel 85, an aqueous solution phase containing liposomes suspended in an aqueous solution and an aqueous solution phase containing fluorescent beads suspended therein are mixed through the annular structure 86 and sent downstream. Such an aqueous solution merges with the oil being sent through the additional oil-phase channel and forms water droplets in oil by passing through the shallow regions 92 and the additional annular structure.

[0076] After forming the water droplets in oil, the portion of the further annular structure (swirl flow path) was observed under a microscope in bright field and fluorescence field. The results are shown in Fig. 12. Fig. 12(a) is an image diagram of the experiment including the appearance of the microchannel 110 used in the test. Fig. 12(b) is an image diagram of the coalesced water droplets remaining at one corner of the hexagonal shape of the swirl flow path. The water droplets in oil are circled. The enlarged images of these water droplets in oil are shown in Figs. 12(c) to (f). Fig. 12(c) is an image diagram when observing the same part as Fig. 12(b) in bright field. Well-formed water droplets in oil were observed. Fig. 12(d) is an image diagram of fluorescence observation of the same part in a non-excited state. The outline of the water droplets in oil seems to be thin and tend to occur. Fig. 12(e) is an image diagram of observing fluorescent beads that produce green fluorescence in an excited state. As is clear from this figure, the fluorescent beads contained in the water droplets in oil could be observed. Fig. 12(f) is an image diagram of observing rhodamine liposomes that produce red fluorescence. It was found that rhodamine was dispersed throughout the water droplets in oil. From these results, it was confirmed that the microchannel system including the microchannel 110 can simultaneously encapsulate fluorescent beads (4.5 μmφ) mimicking cells and rhodamine mimicking liposomes in the water droplets in oil.

[0077] Example 3 Encapsulation test of artificial cells into water droplets in oil The same microchannel system 80 used in the test of Example 1 was prepared. As artificial cells, GUVs with a lipid composition of 100% DOPC were fabricated. A 100 mM aqueous solution of sucrose was used as the inner solution. The artificial cells were suspended in a 100 mM aqueous solution of glucose. The aqueous solution in which the artificial cells were suspended was fed into the flow path 85 together with fluorinate as the oil phase as the aqueous phase. The feeding was performed by pulling the plunger of the syringe 91 attached to the downstream opening to suck from the upstream opening into the flow path 85. After the flow path 85 was filled with liquid, a corner of the annular structure 86 (swirl flow path) was observed under a microscope. The results are shown in Fig. 13. Figs. 13(a) and (b) are image diagrams of water droplets formed and existing at different positions, respectively. The GUV lipid composition (artificial cell) in the water droplet was indicated by an arrow. As a result, it became clear that the microchannel system can encapsulate artificial cells in water droplets in oil.

[0078] Example 4 Encapsulation test of artificial cells in water droplets in oil As artificial cells, GUVs with a lipid composition of 99.9% DOPC and 0.1% rhodamine (Rho-PE) were fabricated. A 200 mM aqueous solution of sucrose was used as the inner solution. The artificial cells were suspended in a 200 mM aqueous solution of glucose. The aqueous solution in which the artificial cells were suspended was fed into the flow path 85 together with fluorinate as the oil phase as the aqueous phase. The test was conducted in the same manner as in Example 3. The results are shown in Fig. 14. When a corner of the annular structure 86 (swirl flow path) was observed under a microscope, water droplets in oil were observed both in the case of non-fluorescent excitation (Fig. 14(a)) and in the case of fluorescent excitation (Fig. 14(b)). When fluorescence was observed, fluorescence generated from rhodamine could be observed. As a result, it became clear that the microchannel system can encapsulate artificial cells in water droplets in oil.

[0079] From the above results, it became clear that the microchannel system can include cells and / or liposomes in water-in-oil droplets. It was suggested that by using such a microchannel system, it is possible to uniformly introduce an activator into cells. It was also suggested that multiple activators can be uniformly introduced into cells thereby. Thereby, it was suggested that it is possible to provide a technique for improving the reaction efficiency, uniformly introducing an activator into material cells, and producing homogeneous target cells.

[0080] Examples of further embodiments are described below. [1] A method for producing target cells including continuously introducing an activator into material cells, forming a first water-in-oil droplet containing the material cells by mixing an aqueous solution containing the material cells and an oil phase, forming a second water-in-oil droplet containing liposomes by mixing an aqueous liposome solution encapsulating the activator and an oil phase, fusing the formed first water-in-oil droplet and the second water-in-oil droplet, and introducing the activator into the material cells within the fused water droplet. A method including the above. [2] A method for producing target cells including introducing an activator into material cells, (a) Mixing an aqueous liposome solution phase encapsulating the activator to be introduced into the material cells and an oil phase at a constant ratio and at a constant rate to continuously form a first water-in-oil droplet containing the liposomes, (b) Mixing an aqueous solution phase containing the material cells and an oil phase to continuously form a second water-in-oil droplet containing the material cells, (c) Sequentially fusing the continuously formed first water-in-oil droplets and the continuously formed second water-in-oil droplets to continuously obtain a third water-in-oil droplet, and (d) Continuously introducing the activator into the material cells within the continuously obtained third water-in-oil droplet to continuously obtain the target cells. A method including the above. [3] A method for producing target cells, comprising sequentially introducing a first activator and a second activator into the material cells, (a’) Mixing a first aqueous liposome phase containing a first activator to be introduced into the material cells in the first stage with an oil phase to continuously form first water-in-oil droplets containing the liposomes, (b’) Mixing an aqueous phase containing the material cells and an oil phase at a certain ratio and at a certain rate to continuously form second water-in-oil droplets containing the material cells, (c’) Sequentially fusing the continuously formed first water-in-oil droplets and the continuously formed second water-in-oil droplets to continuously obtain third water-in-oil droplets, (d’) In the continuously obtained third water-in-oil droplets, the activator is sequentially introduced into the material cells to continuously obtain the material cells in the second stage, (e) Mixing a second aqueous liposome phase containing a second activator to be introduced into the material cells with an oil phase at a certain ratio and at a certain rate to continuously form fourth water-in-oil droplets containing the liposomes, (f) Sequentially fusing the third water-in-oil droplets containing the material cells in the continuously obtained second stage and the continuously formed fourth water-in-oil droplets to continuously obtain fifth water-in-oil droplets, and (g) In the continuously obtained fifth water-in-oil droplets, the second activator is sequentially introduced into the material cells in the second stage to continuously obtain target cells The method comprising the above steps. [4] A method for producing target cells, comprising sequentially introducing all of the first to nth activators (where n is an integer of 3 or more) into the material cells, (a’’) Mixing a first aqueous liposome phase containing a first activator to be introduced into the material cells in the first stage with an oil phase to continuously form first water-in-oil droplets containing the liposomes, (b’’) Mixing an aqueous phase containing the material cells in the first stage and an oil phase at a certain ratio and at a certain rate to continuously form second water-in-oil droplets containing the material cells, (c'') successively fusing the continuously formed first water-in-oil droplets and the continuously formed second water-in-oil droplets to successively obtain third water-in-oil droplets; (d'') successively introducing the activator into the material cells within the continuously obtained third water-in-oil droplets to successively obtain the material cells of the second stage; (e') mixing an n-1th liposome aqueous solution phase encapsulating the n-1th activator to be introduced into the material cells with an oil phase to continuously form (n + 1)th water-in-oil droplets containing the liposomes; (f') successively fusing the nth water-in-oil droplets containing the material cells of the (n - 1)th stage obtained successively and the (n + 1)th water-in-oil droplets formed successively to successively obtain (n + 2)th water-in-oil droplets; (g') successively introducing the n-1th activator into the material cells of the (n - 1)th stage within the continuously obtained (n + 2)th water-in-oil droplets to successively obtain the material cells of the nth stage; (h) mixing an nth liposome aqueous solution phase encapsulating the nth activator to be introduced into the material cells with an oil phase to continuously form (n + 2)th water-in-oil droplets containing the liposomes; (i) successively fusing the (n + 1)th water-in-oil droplets containing the material cells of the nth stage obtained successively and the (n + 2)th water-in-oil droplets formed successively to successively obtain (n + 3)th water-in-oil droplets, and (j) successively introducing the nth activator into the material cells of the nth stage within the continuously obtained (n + 3)th water-in-oil droplets to successively obtain the target cells comprising, wherein the steps (e) to (j) are similarly repeated n - 1 times, and the second to nth activators are successively introduced into the material cells of the second to nth stages respectively. [5] The method according to any one of [1] to [4], wherein the activator is a gene to be introduced. [6] The method according to any one of [1] to [5], wherein the fusion of the first water-in-oil droplets and the second water-in-oil droplets is performed at a certain ratio, for example, 1:1, and / or at a certain speed. [7] The method according to any one of [1] to [6], wherein the aqueous liposome solution phase and the aqueous solution phase containing the material cells each contain a surfactant. [8] The method according to any one of [1] to [7], wherein the lipid membrane constituting the liposome contains FFT10 and / or FFT20 represented by the following chemical formula. [9] The method according to any one of [1] to [8], which is carried out in a microchannel.

[10] The method according to any one of [1] to [9], wherein the fusion includes applying external energy selected from at least one of electricity, temperature, magnetism, light, and ultrasonic waves to promote the fusion.

[11] The method according to any one of [1] to

[10] , which includes adjusting environmental conditions selected from at least one of time, temperature, and gas concentration to promote the introduction of the activator into the cells.

[12] The method according to any one of [1] to

[11] , further comprising a mechanism for enhancing the efficiency of the recovery.

[13] The method according to any one of [1] to

[12] , wherein the mixing of the aqueous solution phase and the oil phase is carried out by the confluence of the microchannels containing them, and the two types of water-in-oil droplets are brought into contact by the confluence of the microchannels containing them.

[14] A microfluidic system for carrying out the method according to any one of [1] to

[13] in a continuous space.

[15] The system according to

[14] , comprising a swirl flow path for producing the water-in-oil droplets.

[16] A microchannel system for producing target cells by introducing an activator into material cells, A first water-in-oil droplet forming flow path for preparing a first water-in-oil droplet containing a material cell suspension in an aqueous solution phase, comprising a first oil phase supply flow path and a first aqueous solution phase supply flow path that merges therewith, A second water-in-oil droplet forming flow path for preparing a second water-in-oil droplet containing a first liposome encapsulating a first activator in an aqueous solution phase, comprising a second oil phase supply flow path and a second aqueous solution phase supply flow path that merges therewith, It is connected so as to merge the first water-in-oil droplet formation flow path and the second water-in-oil droplet formation flow path, and has a fusion part for fusing the first water-in-oil droplet and the second water-in-oil droplet, It is connected to the fusion part, and has an introduction part for bringing the first liposome into contact with the material cell and introducing the activator into the material cell, An opening for discharging the target cell obtained by the introduction A system comprising

[17] A microchannel system for introducing first and second activators into a material cell to produce a target cell, A first water-in-oil droplet formation flow path for preparing a first water-in-oil droplet containing a cell suspension of the material cell in the first stage, comprising a first oil phase supply flow path and a first aqueous solution phase supply flow path merging thereinto, A second water-in-oil droplet formation flow path for preparing a second water-in-oil droplet containing a first liposome encapsulating a first activator in an aqueous solution phase, comprising a second oil phase supply flow path and a second aqueous solution phase supply flow path merging thereinto, It is connected so as to merge the first water-in-oil droplet formation flow path and the second water-in-oil droplet formation flow path, and has a first fusion part for fusing the first water-in-oil droplet and the second water-in-oil droplet to obtain a first fused droplet, It is connected to the first fusion part, brings the first liposome into contact with the material cell, introduces the activator into the material cell, and has a first activator introduction part for obtaining the material cell in the second stage, A third water-in-oil droplet formation flow path for preparing a third water-in-oil droplet containing a second liposome encapsulating a second activator in an aqueous solution phase, comprising a third oil phase supply flow path and a third aqueous solution phase supply flow path merging thereinto, It is connected so as to merge the first activator introduction part and the third water-in-oil droplet formation flow path, and has a second fusion part for fusing the first fused droplet and the third water-in-oil droplet to obtain a second fused droplet, It is connected to the second fusion part, brings the second liposome into contact with the material cell in the second stage, and introduces the second activator into the material cell in the second stage, an opening for discharging the target cells obtained by the introduction A system comprising

[18] A microfluidic system for introducing first to nth activators into material cells (where n is an integer of 3 or more) to produce target cells, a first oil-in-water droplet formation channel for preparing first oil-in-water droplets containing a cell suspension of the material cells in the first stage, the first oil-in-water droplet formation channel comprising a first oil phase supply channel and a first aqueous phase supply channel that merges therewith; a second oil-in-water droplet formation channel for preparing second oil-in-water droplets containing first liposomes encapsulating a first activator in an aqueous phase, the second oil-in-water droplet formation channel comprising a second oil phase supply channel and a second aqueous phase supply channel that merges therewith; a first fusion part that is connected so as to merge the first oil-in-water droplet formation channel and the second oil-in-water droplet formation channel to fuse the first oil-in-water droplets and the second oil-in-water droplets to obtain first fused droplets; a first activator introduction part that is connected to the first fusion part, brings the first liposomes into contact with the material cells, introduces the activator into the material cells, and obtains the material cells in the second stage; third to (n + 1)th oil-in-water droplet formation channels for preparing third to (n + 1)th oil-in-water droplets respectively containing second to nth liposomes encapsulating second to nth activators respectively in an aqueous phase, the third to (n + 1)th oil-in-water droplet formation channels each comprising a third to (n + 1)th oil phase supply channel and a third to (n + 1)th aqueous phase supply channel that merges therewith respectively; second to nth fusion parts that are connected so as to merge the first to (n - 1)th activator introduction parts and the third to (n + 1)th oil-in-water droplet formation channels respectively to obtain second to nth fused droplets for fusing the first to (n - 1)th fused droplets and the third to (n + 1)th oil-in-water droplets respectively; an introduction part that is connected to the second to nth fusion parts, brings the second to nth liposomes into contact with the material cells in the second to nth stages respectively, and introduces the second to nth activators into the material cells in the second to nth stages; an opening for discharging the target cells obtained by the introduction A system comprising

[19] The system according to any one of

[14] to

[18] , further comprising a control unit for controlling the flow rate and / or flow velocity in the system.

[20] The system according to any one of

[14] to

[19] , wherein the fusion part and / or the introduction part comprises an electrical stimulation mechanism, a shape difference from the flow paths before and after it, a heating mechanism and / or a magnetic field generation mechanism.

[21] The system according to any one of

[14] to

[20] , further comprising a timing control unit for controlling the timing of the movement of the water droplets in oil.

[22] The system according to any one of

[14] to

[21] , further comprising a temperature management mechanism for managing the temperature in the system.

[23] The system according to any one of

[14] to

[23] , wherein the fusion part and / or the introduction part comprises an annular flow path and / or a swirl flow path.

[0081] Although some embodiments of the present invention have been described, these embodiments are presented by way of example and are not intended to limit the scope of the invention. These novel embodiments can be implemented in various other forms, and various omissions, replacements, and changes can be made without departing from the gist of the invention. These embodiments and their modifications are included in the scope and gist of the invention, and are included in the invention described in the claims and its equivalent scope.

Explanation of reference numerals

[0082] 1, 1b, 1n... material cells, 2, 2b, 2n... liposomes, 3... target cells (or material cells at a specific stage), 4... target cells, 10, 30, 50, 80, 110... microchannel systems, 11... main trunk, 12... branch, 13, 14, 14a, 14b, 14n... water-in-oil droplet formation channels, 15, 20... oil phase supply channels, 16, 21... aqueous solution supply channels, 17... oil phase, 18... cell-containing solution (aqueous solution), 19, 24, 24b, 24n, 95, 95a, 95b, 95c, 95d... water-in-oil droplets, 20b, 20n-1... oil phase channels, 21... aqueous solution phase channels, 25... fused droplets, 22... liposome-containing solution (aqueous solution), 31, 32... droplet generation parts, 33... confluence part, 34, 34a, 34b... droplet fusion parts, 35, 35a, 35b... activator introduction parts (for example, gene introduction parts), 52... main trunk channel, 53... stimulation mechanism, a, n... activators, 81... substrate, 83... lid, 81... syringe, 82... groove, 84... opening, 85, 111... channels, 86... annular structure (an example of a swirl channel), 87, 87a, 87b, 87c... tubes, 88, 89... containers, 90... joint, 92... shallow region

Claims

1. A method for producing target cells, comprising introducing an activator into material cells, forming first water-in-oil droplets containing the material cells by mixing an aqueous solution containing the material cells with an oil phase, forming second water-in-oil droplets containing liposomes by mixing an aqueous liposome solution encapsulating the activator with an oil phase, fusing the formed first water-in-oil droplets and the second water-in-oil droplets to form fused droplets, and introducing the activator into the material cells within the fused droplets The method comprising the steps of.

2. A method for producing target cells, comprising introducing an activator into material cells, (a) intermittently mixing an aqueous liposome solution phase encapsulating the activator to be introduced into the material cells with an oil phase to continuously form first water-in-oil droplets containing the liposomes, (b) intermittently mixing an aqueous solution phase containing the material cells with an oil phase to continuously form second water-in-oil droplets containing the material cells, (c) sequentially fusing the continuously formed first water-in-oil droplets and the continuously formed second water-in-oil droplets to continuously obtain third water-in-oil droplets, and (d) introducing the activator into the material cells within each of the continuously obtained third water-in-oil droplets to continuously obtain the target cells The method comprising the steps of.

3. A method for producing target cells, comprising introducing first and second activators into material cells, (a') mixing an aqueous solution phase of first liposomes encapsulating the first activator to be introduced into the material cells in the first stage with an oil phase at a certain ratio and at a certain speed to continuously form first water-in-oil droplets containing the liposomes, (b') mixing an aqueous solution phase containing the material cells with an oil phase to continuously form second water-in-oil droplets containing the material cells, (c') sequentially fusing the continuously formed first water-in-oil droplets and the continuously formed second water-in-oil droplets to continuously obtain third water-in-oil droplets, (d') introducing the activator into the material cells within each of the continuously obtained third water-in-oil droplets in sequence to continuously obtain the material cells in the second stage, (e) mixing an aqueous solution phase of second liposomes encapsulating the second activator to be introduced into the material cells with an oil phase to continuously form fourth water-in-oil droplets containing the liposomes, (f) successively fusing the third water-in-oil droplets containing the material cells of the second stage obtained continuously and the fourth water-in-oil droplets formed continuously to successively obtain fifth water-in-oil droplets, and (g) introducing the second activator into the material cells of the second stage in each of the successively obtained fifth water-in-oil droplets to successively obtain target cells A method comprising the steps of: [

4. ] A method for producing target cells, comprising successively introducing all of the first to nth activators (where n is an integer of 3 or more) into material cells, (a'') mixing a first liposome aqueous phase encapsulating a first activator to be introduced into the material cells of the first stage and an oil phase at a constant ratio and at a constant rate to continuously form first water-in-oil droplets containing the liposomes, (b'') mixing an aqueous phase containing the material cells of the first stage and an oil phase at a constant ratio and at a constant rate to continuously form second water-in-oil droplets containing the material cells, (c'') successively fusing the first water-in-oil droplets formed continuously and the second water-in-oil droplets formed continuously to successively obtain third water-in-oil droplets, (d'') introducing the activator into the material cells in the successively obtained third water-in-oil droplets in order to successively obtain the material cells of the second stage, (e') mixing an (n-1)th liposome aqueous phase encapsulating an (n-1)th activator to be introduced into the material cells and an oil phase at a constant ratio and at a constant rate to continuously form (n+1)th water-in-oil droplets containing the liposomes, (f') successively fusing the nth water-in-oil droplets containing the material cells of the (n-1)th stage obtained continuously and the (n+1)th water-in-oil droplets formed continuously to successively obtain (n+2)th water-in-oil droplets, (g') introducing the (n-1)th activator into the material cells of the (n-1)th stage in the successively obtained (n+2)th water-in-oil droplets in order to successively obtain the material cells of the nth stage, (h) mixing an nth liposome aqueous phase encapsulating an nth activator to be introduced into the material cells and an oil phase at a constant ratio and at a constant rate to continuously form (n+2)th water-in-oil droplets containing the liposomes, (i) sequentially fusing the (n + 1)-th water-in-oil droplet containing the material cells of the n-th stage obtained continuously and the (n + 2)-th water-in-oil droplet formed continuously to continuously obtain the (n + 3)-th water-in-oil droplet, and (j) within the continuously obtained (n + 3)-th water-in-oil droplet, the n-th activator is sequentially introduced into the material cells of the n-th stage to continuously obtain the target cells The method according to claim 1, wherein the steps (e) to (j) are similarly repeated n - 1 times, and the second to n-th activators are sequentially introduced into the material cells of the second to n-th stages, respectively.

5. The method according to any one of claims 1 to 4, wherein the activator is a gene.

6. The method according to any one of claims 1 to 4, wherein the fusion between the water-in-oil droplets is performed at a ratio of 1:

1.

7. The method according to any one of claims 1 to 4, wherein the oil layer for encapsulating the liposome aqueous phase and the aqueous phase containing the material cells contains a surfactant.

8. The method according to any one of claims 1 to 4, wherein the lipid membrane constituting the liposome contains FFT10 and / or FFT20 represented by the following chemical formula.

9. The method according to any one of claims 1 to 4, which is performed in a microchannel.

10. The method according to any one of claims 1 to 4, wherein the mixing of the aqueous phase and the oil phase and / or the fusion of the water-in-oil droplets are performed by the confluence of the respective microchannels containing them and / or the swirl channel after the confluence.

11. A microchannel system for producing target cells by introducing an activator into material cells, comprising: a first water-in-oil droplet forming channel for preparing a first water-in-oil droplet containing a material cell suspension in an aqueous phase, comprising a first oil phase supply channel and a first aqueous phase supply channel that merges therewith; a second water-in-oil droplet forming channel for preparing a second water-in-oil droplet containing a first liposome encapsulating a first activator in an aqueous phase, comprising a second oil phase supply channel and a second aqueous phase supply channel that merges therewith; connected so as to merge the first water-in-oil droplet forming channel and the second water-in-oil droplet forming channel, and a fusion part for fusing the first water-in-oil droplet and the second water-in-oil droplet; connected to the fusion part, a introducing part for bringing the first liposome into contact with the material cells and introducing the activator into the material cells; and an opening for discharging the target cells obtained by the introduction. A system comprising.

12. A microchannel system for introducing first and second activators into material cells to produce target cells, A first water-in-oil droplet formation channel for preparing a first water-in-oil droplet containing a cell suspension of material cells in a first stage, the first water-in-oil droplet formation channel comprising a first oil phase supply channel and a first aqueous phase supply channel that merges therewith, A second water-in-oil droplet formation channel for preparing a second water-in-oil droplet containing a first liposome encapsulating a first activator in an aqueous phase, the second water-in-oil droplet formation channel comprising a second oil phase supply channel and a second aqueous phase supply channel that merges therewith, A first fusion section that is connected so as to merge the first water-in-oil droplet formation channel and the second water-in-oil droplet formation channel, and that fuses the first water-in-oil droplet and the second water-in-oil droplet to obtain a first fused droplet, A first activator introduction section that is connected to the first fusion section, contacts the first liposome and the material cells, and introduces the activator into the material cells to obtain the material cells in a second stage, A third water-in-oil droplet formation channel for preparing a third water-in-oil droplet containing a second liposome encapsulating a second activator in an aqueous phase, the third water-in-oil droplet formation channel comprising a third oil phase supply channel and a third aqueous phase supply channel that merges therewith, A second fusion section that is connected so as to merge the first activator introduction section and the third water-in-oil droplet formation channel, and that obtains a second fused droplet for fusing the first fused droplet and the third water-in-oil droplet, An introduction section that is connected to the second fusion section, contacts the second liposome and the material cells in the second stage, and introduces the second activator into the material cells in the second stage, An opening for discharging the target cells obtained by the introduction, And a system comprising the same.

13. A microchannel system for introducing first to nth activators into material cells (where n is an integer of 3 or more) to produce target cells, A first water-in-oil droplet formation channel for preparing a first water-in-oil droplet containing a cell suspension of material cells in a first stage, the first water-in-oil droplet formation channel comprising a first oil phase supply channel and a first aqueous phase supply channel that merges therewith, A second water-in-oil droplet formation channel for preparing a second water-in-oil droplet containing a first liposome encapsulating a first activator in an aqueous phase, the second water-in-oil droplet formation channel comprising a second oil phase supply channel and a second aqueous phase supply channel that merges therewith, It is connected so as to merge the first water-in-oil droplet formation flow path and the second water-in-oil droplet formation flow path, and fuses the first water-in-oil droplet and the second water-in-oil droplet to obtain a first fused droplet, and a first fusion part for It is connected to the first fusion part, brings the first liposome into contact with the material cell, introduces the activator into the material cell, and a first activator introduction part for obtaining the material cell at the second stage. Third to (n + 1)th oil phase supply flow paths, and third to (n + 1)th aqueous solution phase supply flow paths respectively merging thereinto, and third to (n + 1)th water-in-oil droplets each containing second to nth liposomes each encapsulating second to nth activators in the aqueous solution phase, respectively. Third to (n + 1)th water-in-oil droplet formation flow paths for preparing It is connected so as to merge the first to (n - 1)th activator introduction parts and the third to (n + 1)th water-in-oil droplet formation flow paths, and the first to (n - 1)th fused droplets and the third to (n + 1)th water-in-oil droplets, respectively. Second to nth fusion parts for obtaining second to nth fused droplets for fusing respectively It is connected to the second to nth fusion parts, brings the second to nth liposomes into contact with the material cells at the second to nth stages respectively, and an introduction part for introducing the second to nth activators into the material cells at the second to nth stages. An opening for discharging the target cells obtained by the introduction A system comprising

14. The system according to any one of claims 11 to 13, further comprising a control part for controlling the flow rate and / or flow velocity in the system.

15. The system according to any one of claims 11 to 13, wherein the fusion part and / or the introduction part comprises an electrical stimulation mechanism, a shape difference from the flow paths before and after it, a heating mechanism and / or a magnetic field generation mechanism.

16. The system according to any one of claims 11 to 13, further comprising a timing control part for controlling the timing of movement of the water-in-oil droplets.

17. The system according to any one of claims 11 to 13, further comprising a temperature management mechanism for managing the temperature in the system.

18. The system according to any one of claims 11 to 13, wherein the fusion part and / or the introduction part comprises an annular flow path and / or a swirl flow path.

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