Patent Description:
In recent years, regenerative medicine/cell therapy is actively researched and there is a growing need for a flow cytometer as a technique to quickly evaluate a cell. The flow cytometer is an analysis technique in which microparticles to be analyzed are poured into a fluid in an aligned state, and the microparticles are analyzed and sorted by irradiating the microparticles with laser light or the like and detecting fluorescence and scattered light emitted from each of the microparticles, and the flow cytometer is used as a tool to analyze a cell in the research on the regenerative medicine/cell therapy. As a device of the above-described flow cytometer, for example, a microparticle measurement devices as disclosed in Patent Documents <NUM> to <NUM> are known.

Conventionally, it is known that in a case of feeding cell sample liquid without stirring the cell sample liquid at the time of the feeding, clogging easily occurs inside a tube because the high-concentration liquid is fed, and stability of a device to which the cell sample liquid is supplied is impaired. Additionally, particularly at the time of liquid feeding to a microparticle measurement device, it is also known that sorting processing cannot be executed in time and a cell sample is wasted because the high-concentration cell sample liquid flows in.

On the other hand, in a case of closed-type liquid feeding using a conventional bag, liquid is fed after stirring the liquid by using a stirring method such as shaking the bag or stirring the liquid with a stirrer. However, a rocking shaker or the like requires a space and cost. Additionally, a magnet stirrer can have a size more reduced than in the case of shaking by rocking, but there may be a risk that a stirrer rotor blade damages a cell sample. Furthermore, it is also possible to consider a stirring method by generating a reflux flow inside the bag, but a pump that generates the reflux flow is necessary, and therefore, upsizing of the device and cost increase are estimated. Thus, while stirring is indispensable for stable liquid feeding to the microparticle measurement device, it is desired to eliminate the stirring as much as possible in terms of device development.

Here, a general cell sample liquid feeding bag has a quadrangular shape, a liquid inlet/outlet port has a large tube diameter, and there is a problem of air mixture when the cell sample liquid inside the bag is sucked out, and also there is a problem that a dead volume is generated due to the liquid remaining at a corner of the bag. Additionally, the air mixture is a phenomenon desired to be prevented in feeding the cell sample liquid to the microparticle measurement device because the air mixture becomes a serious disturbance at the time acquiring a signal of a cell particle. Furthermore, it is necessary to reduce the dead volume as much as possible because samples are wasted.

<CIT> discloses a liquid suction tool, liquid supply unit and an automated analyzer that can reduce an amount of the liquid remaining in the suction portion while maintaining the strength of the suction portion. The liquid suction tool has a rod-like suction portion and a connecting member. The suction portion is inserted into the storage bag. A groove portion is formed on a side surface of the suction portion to pass the liquid.

<CIT> discloses an inflatable bioreactor bag for cell cultivation, comprising a top and a bottom sheet of flexible material, joined together to form two end edges and two side edges, wherein one baffle or a plurality of baffles extend from the bottom sheet in a region where the shortest distance to any one of the two end edges is higher than about one fourth of the shortest distance between the two end edges.

<CIT> discloses a mixing bag having an interior mixing chamber having a volume sufficient to receive a predetermined weight of substantially dry material and a selected volume of process compatible solution to form a solution having the material in suspension with a selected concentration. The mixing bag includes at least two sealable openings. One opening is configured for at least one of enabling transporting the substantially dry material into the interior mixing chamber, enabling agitation of the material and process compatible solution in the interior mixing chamber to form the solution and enabling withdrawal of the solution at the selected concentration. The other opening is configured for at least one of injection of a process compatible solution into the interior mixing chamber to form the solution, enabling agitation of the material and process compatible solution in the interior mixing chamber and enabling withdrawal of the solution.

<CIT> aims to obtain an antithrombotic polymer composition by mixing a polyamide with a specific polyalkyl ether / polyaryl ether sulfone-based copolymer. This composition comprises (A) <NUM>-<NUM> pts. of an polyalkyl ether / polyaryl ether sulfone copolymer which is composed of repeating units of (i) the formula -Ar<NUM>-X-Ar<NUM>-O-, (ii) the formula -Ar<NUM>-Y-Ar<NUM>- and (iii) the formula (-RO-)k, [X is SO2; Ar<NUM> to Ar<NUM> are each a (nucleus-substituted) <NUM>-30C bifunctional aromatic hydrocarbon or the like; Y is a (substituted) <NUM>-13C bifunctional aromatic hydrocarbon or the like; R is a <NUM>-3C alkylene; and k is a number of repeating unit to make the molecular weight of the polyoxyalkylene structure of (RO)k have <NUM>,<NUM>-<NUM>,<NUM>] and has the content of the repeating unit of the formula iii to the total of the repeating units of the formula (i) to the formula (iii) of <NUM>-<NUM> wt. % by weight ratio and >=<NUM> reduced viscosity measured in a mixed solvent of phenol/<NUM>,<NUM>,<NUM>,<NUM>-tetrachloroethane of <NUM>/<NUM> (weight ratio) in <NUM>/dl concentration at <NUM> and (B) <NUM>-<NUM> pts. of a polyamide.

<CIT> aims to provide a method for stably introducing a high-quality hydrophilic layer capable of sufficiently inhibiting nonspecific adsorption of a bio-related substance such as protein into a plastic surface of a substrate, and provides a production method of a substrate having a hydrophilic layer on a surface thereof, which includes: a substrate preparation step of preparing a substrate having a plastic surface; a plasma treatment step <NUM> of exposing the plastic surface of the substrate to plasma containing rare gas; a plasma treatment step <NUM> of exposing the plastic surface of the substrate to plasma containing fluorocarbon gas; a primer layer formation step of forming a primer layer containing polysiloxane by polymerizing a silanol compound having an organic group that contains a carbon atom directly bonded to a silicon atom and has a functional group, on the surface of the substrate exposed to plasma; and a hydrophilic layer formation step of linking the primer layer with a hydrophilic polymer via a covalent bond by the reaction of the functional group on a side chain of the polysiloxane with a functional group of the hydrophilic polymer.

Considering the above-described situation, it is desired to develop a cell sample liquid feeding bag having a new shape, a feeding method thereof, and a feeding device thereof which enable stable liquid feeding.

Therefore, the present technology is mainly directed to providing a technology that enables stable liquid feeding.

The problems are solved by the subject matter of the independent claims.

The present technology first provides a cell sample liquid feeding bag including at least: an outflow port from which cell sample liquid flows out; a bottom portion including a reservoir capable of storing cells, and at least partly including a slope; and a first inner tube extending from the outflow port toward the reservoir up to a position not contacting the reservoir, in which the cell sample liquid is fed from a reservoir side toward an outflow port side of the first inner tube.

In the cell sample liquid feeding bag according to the present technology, a cross-sectional shape of the bottom portion has a substantially V-shape.

Additionally, the cell sample liquid feeding bag according to the present technology may further include an outer tube extending from the outflow port toward outside of the bag, and the outer tube may include at least two or more branched paths.

Furthermore, the cell sample liquid feeding bag according to the present technology may further include at least one or more ports besides the outflow port. In this case, the ports may be provided at a bag wall surface. Additionally, in this case, one of the ports may further include a second inner tube extending toward the inside of the bag, and the second inner tube may be used for cell sample liquid injection. Furthermore, in this case, an inner diameter of the second inner tube may be larger than an inner diameter of the first inner tube.

Additionally, the cell sample liquid feeding bag according to the present technology may have at least a part of a bag inner wall applied with a coating that inhibits nonspecific adsorption of the sample. In this case, the coating may include one kind selected from a group consisting of low molecular protein, silicon, and a water-soluble polymer. Additionally, in this case, the low molecular protein may include albumin, and the water-soluble polymer may include at least one or more kinds selected from a group consisting of casein, gelatin, dextran, polyacrylamide, polyvinyl alcohol, polyvinylpyrrolidone, and polyethylene glycol.

Furthermore, the cell sample liquid feeding bag according to the present technology may further include a support portion that supports an upright posture of the bag.

Additionally, the present technology also provides a cell sample liquid feeding method using a cell sample liquid feeding bag according to claim <NUM> and the method at least includes: an ejecting step of ejecting liquid toward the reservoir before starting cell sample liquid feeding.

In the cell sample liquid feeding method according to the present technology, the cell sample liquid feeding bag may further include an outer tube extending from the outflow port toward the outside of the bag, and the ejection may be executed by reverse rotation of an outer tube pump.

Furthermore, the present technology also provides a cell sample liquid feeding device including at least a cell sample liquid feeding bag including at least a cell sample liquid feeding bag according to claim <NUM>.

The cell sample liquid feeding device according to the present technology may further include an ejection mechanism capable of ejecting liquid toward the reservoir before feeding the cell sample liquid. In this case, the cell sample liquid feeding bag further includes an outer tube extending from the outflow port toward the outside of the bag, and the ejection mechanism is run by reverse rotation of an outer tube pump.

Furthermore, the present technology also provides a microparticle measurement device including at least the cell sample liquid feeding device according to the present technology.

In the present technology, the "microparticle" broadly includes, for example: biologically relevant microparticles such as cells, microbes, and liposomes; synthetic particles such as a latex particle, a gel particle, and a particle for an industrial use; and the like.

Additionally, the biologically relevant microparticles include a chromosome, a liposome, a mitochondrion, an organelle (cell organ), and the like constituting various kinds of cells. The cells include animal cells (such as hematopoietic cell) and plant cells. The microbes include: bacteria such as coli bacilli; viruses such as tobacco mosaic viruses; fungi such as yeast; and the like. Additionally, the biologically relevant microparticles also include biologically relevant polymers such as nucleic acids, proteins, and a complex thereof. Additionally, the particle for the industrial use may include, for example, an organic or inorganic polymer material, a metal, or the like. The organic polymer materials include polystyrene, styrenedivinylbenzene, polymethyl methacrylate, and the like. The inorganic polymer materials include glass, silica, a magnetic material, and the like. The metal includes gold colloid, aluminum, and the like. Generally, these microparticles each have a spherical shape, but in the present technology, the shape may be non-spherical, and a size, mass, and the like thereof are not particularly limited.

According to the present technology, the liquid can be stably fed. Note that the effect recited herein is not necessarily limited and may be any one of those recited in the present disclosure.

Preferred embodiments to carry out the present technology will be described below with reference to the drawings. Note that the embodiments described below illustrate examples of the representative embodiments of the present technology and the scope of the present technology should not be interpreted to be limited by these embodiments. Note that the description will be provided in the following order.

<FIG> is a schematic view illustrating a first embodiment of a cell sample liquid feeding bag <NUM> according to the present technology. The cell sample liquid feeding bag <NUM> according to the present technology includes at least: an outflow port <NUM> from which cell sample liquid flows out; a reservoir <NUM> capable of storing cells; a bottom portion <NUM> at least partly including a slope; and a first inner tube <NUM> extending from the outflow port <NUM> toward the reservoir <NUM> up to a position not contacting the reservoir <NUM>. Additionally, the cell sample liquid is fed from the reservoir <NUM> side toward the outflow port <NUM> side of the first inner tube <NUM>.

Conventionally, shake stirring by rocking, or the like is executed as a method of stirring cells inside a closed-type bag, however; since the cell sample liquid feeding bag <NUM> according to the present technology is used, a cell sample that has settled in the reservoir <NUM> is whirled up and the cell sample can be kept in a stirred state constantly having a uniform concentration without using a stirrer, and as a result, the liquid can be stably fed. For this reason, downsizing of the device and reduction of a manufacturing cost can be achieved. Furthermore, the cell sample inside the bag can be fed while reducing a dead volume.

Additionally, since a conventional cell sample liquid feeding bag adopts a form in which a liquid feeding port is located at a lowermost end in a hung state and liquid is sucked from below, cells are made to settle at the time of liquid feeding without stirring, and furthermore, a sample flow is very slow such as about <NUM>µL/min, and a thin feeding tube (for example, φ <NUM> - <NUM> or the like) is also used, and therefore, there is a high possibility that the tube is clogged in a direction sucking out the liquid downward from the lowermost end before starting the liquid feeding. On the other hand, in the cell sample liquid feeding bag <NUM> according to the present technology, the cell sample liquid is fed from the reservoir <NUM> side toward the outflow port <NUM> side of the first inner tube <NUM>, and therefore, a tube is hung from an uppermost end to a lowermost end and a sucking portion has a shape sucking the liquid with the tube from a lower side to an upper side, and the possibility of clog inside the tube can be reduced before start the feeding.

Furthermore, when the cell sample liquid feeding bag <NUM> according to the present technology is used, it is sufficient that the bag is set upright. Therefore, a degree of freedom relative to a method of fixing the bag to a device is more increased than in the conventional cell sample liquid feeding bag. Additionally, as a method of setting the bag upright, for example, it is possible to exemplify; a method of hanging the bag at a hook or the like; putting the bag in a container or the like and fixing the bag inside the container; or the like. Furthermore, besides the above, there also is a method of providing a support portion <NUM> described later.

A shape of the bottom portion <NUM> is not particularly limited as far as the shape includes the reservoir <NUM> capable of storing the cells, and at least partly includes a slope, and a shape of a second embodiment as illustrated in <FIG> or a shape of a third embodiment illustrated in <FIG> or the like may also be adopted. Since the bottom portion <NUM> has the above-described shape, precipitated cells can be collected in the reservoir <NUM>, and therefore, the cells that are settling can be fed as much as possible without waste.

A cross-sectional shape of the bottom portion <NUM> is not also particularly limited but preferably has a substantially V-shape as illustrated in the first embodiment of <FIG>. With this cross-sectional shape, the precipitated cells can be more efficiently collected in the reservoir <NUM>. Additionally, in this case, an angle (inclination angle) θ (see <FIG>) between a horizontal plane and the cross section of the bottom portion <NUM> is also not particularly limited but is preferably <NUM>° to <NUM>°, more preferably <NUM>° to <NUM>°, and particularly preferably <NUM>° to <NUM>°.

Lengths of D1, D2, and D3 illustrated in <FIG> are also not particularly limited, and for example, D1 can be <NUM>, D2 can be <NUM>, and D3 can be <NUM>. A reason why D1 is set to <NUM> is to secure a margin for making a hole because there may be a case where it becomes necessary to make a hole in an upper portion of the bag when the bag is used in a hung state.

Additionally, in the present technology, a distance between the reservoir <NUM> and the first inner tube <NUM> is not particularly limited and may be, for example, about <NUM>.

Furthermore, in the present technology, an outer diameter, an inner diameter, a material, and the like of the first inner tube <NUM> are also not particularly limited, and for example, a PEEK tube having an outer diameter of <NUM>/<NUM> inches, an inner diameter of <NUM> or <NUM>, or the like can be used as the first inner tube <NUM>.

In the present technology, as illustrated in a fourth embodiment of <FIG>, an outer tube <NUM> extending from the outflow port <NUM> toward the outside of the bag is further provided, and the outer tube <NUM> can include at least two or more branched paths. With the branched paths, it is possible to separately provide a tube used at the time of injecting the cell sample into the bag and a tube used at the time of sucking out the cell sample, and the tube used at the time of injecting the cells can be aseptically cut and closed after the injection, and therefore, a risk of contamination can be reduced and convenience of a user can be improved.

Note that, in the present technology, a shape of the outer tube <NUM> is not limited to a shape illustrated in the fourth embodiment of <FIG>, and may not necessarily include the branched paths as illustrated in a fifth embodiment of <FIG>.

Additionally, in the present technology, an inner diameter, a length, a material, and the like of the outer tube <NUM> are not particularly limited, and for example, a medical tube having an inner diameter of <NUM>, a length of <NUM> to <NUM>, and including polyethylene (PE), polyvinyl chloride (PVC), thermoplastic elastomer (TPE), or the like can be used as the outer tube <NUM>.

Furthermore, in the present technology, as illustrated in the fifth embodiment of <FIG>, at least one or more ports <NUM> can be further provided besides the outflow port <NUM>. With this structure, the port <NUM> can be used, for example, as an injection port at the time of injecting the cell sample into the bag, and as for the port <NUM> used for the injection, a tube connected to the port <NUM> can be aseptically cut and closed after the injection, and therefore reduce the risk of contamination can be reduced and the convenience of a user can be improved.

In a case where the cell sample liquid feeding bag <NUM> according to the present technology includes the port(s) <NUM> as illustrated in the fifth embodiment of <FIG>, a sixth embodiment of <FIG>, and a seventh embodiment of <FIG>, it is preferable that the port(s) <NUM> be provided at a bag wall surface.

Additionally, in a case where the ports <NUM> are provided at the bag wall surface, one of the ports <NUM> further includes a second inner tube <NUM> extending toward the inside of the bag as illustrated in the sixth embodiment of <FIG>, and the second inner tube <NUM> can also be used for cell sample liquid injection. This improves the convenience of a user.

Furthermore, in this case, in the present technology, an inner diameter of the second inner tube <NUM> is preferably larger than the inner diameter of the first inner tube <NUM>. With this large inner diameter, pressure drop is reduced and it becomes easy to feed the cell sample at the time of injecting the cell sample faster than at the time of sucking out the cell sample.

Additionally, as illustrated in the sixth embodiment of <FIG>, in the case where the cell sample liquid feeding bag <NUM> according to the present technology includes the two or more ports <NUM>, one of the ports <NUM> may be used for the cell sample liquid injection, and the other port(s) <NUM> may be used to inject liquid such as reagent into the bag or inject a dyeing solution, a transgenic virus solution, or the like. Furthermore, as for any of the ports <NUM>, it is possible to aseptically cut and close the tube connected to each of the ports <NUM> after the injection, and therefore, the risk of contamination can be reduced or the convenience of a user can be improved.

As illustrated in the seventh embodiment of <FIG>, in the case where the cell sample liquid feeding bag <NUM> according to the present technology includes the two or more ports <NUM>, the number and installation positions thereof are also not particularly limited as far as the ports are provided at the bag wall surface. In the present technology, as illustrated in the seventh embodiment, the plurality of ports <NUM> can be provided on a left side and a right side of the outflow port <NUM> and in the vicinity of a tip of the bottom surface <NUM>. Also, as illustrated in the present embodiment, each second inner tube <NUM> may extend through the inside of each of the ports <NUM> toward the inside of the bag.

In the present technology, at least a part of the bag inner wall can be applied with a coating that inhibits nonspecific adsorption of the sample. It is known that cells generally cause nonspecific adsorption, and therefore, it is assumed that there are cells that nonspecifically adsorb also onto the bag inner wall and fail to fall down to the reservoir <NUM>. Accordingly, by applying the coating in order to reduce the nonspecific adsorption, the cells can be collected, made to settle, it is possible to more efficiently collect the cells and make the cells settle and be accumulated in the reservoir <NUM>. With this effect, the cell sample having a stable concentration can be continuously supplied in a case of executing a cell sample liquid feeding method described later.

In the present technology, the coating is not particularly limited, but it is preferable to use one kind selected from a group consisting of low molecular protein, silicon, and a water-soluble polymer. Additionally, the low molecular protein preferably includes albumin, and the water-soluble polymer includes at least one or more kinds selected from a group consisting of casein, gelatin, dextran, polyacrylamide, polyvinyl alcohol, polyvinylpyrrolidone, and polyethylene glycol.

Also, in the present technology, at least a part of the bag inner wall can also be applied with a coating that promotes specific adsorption of the sample. With this coating, it is possible to trap unnecessary cells and improve purity of necessary cells in a collected solution.

The cell sample liquid feeding bag <NUM> according to the present technology may further include the support portion <NUM> that supports an upright posture of the bag. As described above, the cell sample liquid feeding bag <NUM> according to the present technology is required to be used in the upright state, and as one of methods thereof, it may be conceivable to devise a shape of the bag such that the bag is made to stand on its own by the support portion <NUM>.

The shape of the support portion <NUM> is not particularly limited, but as illustrated in an eighth embodiment of <FIG>, it is preferable that the bottom portion <NUM> of the bag be provided with an outer edge.

<FIG> illustrate a ninth embodiment of a cell sample liquid feeding bag <NUM> according to the present technology. Additionally, A to E of <FIG> are modified examples of the ninth embodiment of the cell sample liquid feeding bag <NUM> according to the present technology. The cell sample liquid feeding bag <NUM> according to the present technology can have a structure in which two transparent flexible films are partly bonded together as illustrated in the ninth embodiment.

<FIG> illustrate a tenth embodiment of a cell sample liquid feeding bag <NUM> according to the present technology. Additionally, A to E of <FIG> are modified examples of the tenth embodiment of the cell sample liquid feeding bag <NUM> according to the present technology. The cell sample liquid feeding bag <NUM> according to the present technology can also formed by using one transparent flexible film as illustrated in the tenth embodiment.

The cell sample liquid feeding bag <NUM> according to the present technology may have a hole used to hang the cell sample liquid feeding bag <NUM> at a hook or the like as illustrated in the ninth embodiment, the tenth embodiment, and the like described above.

A product name when the cell sample liquid feeding bag <NUM> according to the present technology is distributed is not particularly limited, and can be named as a medical bag, a medical soft bag, a blood bag, an infusion bag, a culture bag, a medical container, a medication container, an infusion containers, and the like, for example.

<FIG> is a referential view illustrating an exemplary flow form of the cell sample liquid feeding bag <NUM> according to the present technology. As illustrated in <FIG>, the cell sample liquid feeding bag <NUM> according to the present technology may be distributed as a part of a product, such as a cartridge, a unit, a device, a kit, and an instrument for a closed-type cell sorter. Note that the number of the cell sample liquid feeding bags <NUM> according to the present technology are three in <FIG>, but the number of this products is not particularly limited. Additionally, as illustrated in <FIG>, a waste solution bag, a sheath liquid bag, a microchip, or the like may be individually connected to this product, and the number thereof is also not particularly limited.

In the cell sample liquid feeding method according to the present technology, at least an ejection process using the above-described cell sample liquid feeding bag <NUM> is executed. Note that the description thereof is omitted here because the cell sample liquid feeding bag <NUM> is similar to the one described above.

The ejection process is a process to eject liquid toward the reservoir <NUM> before starting the cell sample liquid feeding. With the execution of the ejection process, the cell sample liquid concentration can be made uniform by whirling up the cell sample that has settled in the reservoir <NUM> by leaving the cell sample liquid feeding bag <NUM> stationary, and stable liquid feeding can be performed. <FIG> is a graph prepared as a drawing and illustrating a result of studying achievement of a uniform cell sample concentration by the ejection process. In <FIG>, a vertical axis represents the cell (sample) concentration (× <NUM><NUM>/mL), and a horizontal axis represents time (sec).

As illustrated in <FIG>, it can be confirmed that feeding of a low-concentration cell sample starts with stirring executed by ejecting the liquid at the beginning of the ejection process, and then the concentration gradually becomes constant. Note that an amount of the liquid ejected in the ejection process is not particularly limited, and for example, a maximum amount can be set, as a guide, to an amount of about <NUM>/<NUM> of a volume of the cell sample liquid feeding bag <NUM>.

<FIG> is a schematic conceptual view schematically illustrating an exemplary embodiment of the ejection process. In the cell sample liquid feeding method according to the present technology, as illustrated in <FIG>, the cell sample liquid feeding bag <NUM> further includes the outer tube <NUM> extending from the outflow port <NUM> toward the outside of the bag, and the ejection can be executed by reverse rotation of an outer tube pump <NUM>. In the embodiment illustrated in <FIG>, the liquid to be ejected is prepared at a tip of the outer tube, and the outer tube pump <NUM> is reversely rotated before starting the cell sample liquid feeding, thereby whirling up the cell sample that has settled in the reservoir <NUM>. With this process, the cell sample concentration can be made uniform as described above, and it is possible to prepare start of the liquid feeding.

In the present technology, other processes may be executed in addition to the above-described ejection process as far as the effects of the present technology are not impaired. The other processes include, for example, methods and the like executed by a fluid control unit <NUM>, a light emission unit <NUM>, a light detection unit <NUM>, an analysis unit <NUM>, a sorting unit <NUM>, an electric charging unit <NUM>, a recording unit <NUM>, and a display unit <NUM>, an input unit <NUM>, a control unit <NUM>, and the like in a microparticle measurement device <NUM> described later.

A cell sample liquid feeding device <NUM> according to the present technology at least includes the above-described cell sample liquid feeding bag <NUM>. Note that the description thereof is omitted here because the cell sample liquid feeding bag <NUM> is similar to the one described above.

The cell sample liquid feeding device <NUM> according to the present technology may further include an ejection mechanism capable of ejecting liquid toward the reservoir <NUM> before feeding the cell sample liquid. Since the ejection mechanism is provided, the cell sample liquid concentration can be made uniform by whirling up the cell sample that has settled in the reservoir <NUM> by leaving the cell sample liquid feeding bag <NUM> stationary, and stable liquid feeding can be performed.

Additionally, in the cell sample liquid feeding device <NUM> according to the present technology, as illustrated in <FIG> described above, the cell sample liquid feeding bag <NUM> further includes the outer tube <NUM> extending from the outflow port toward the outside of the bag, and the ejection mechanism can be run by reverse rotation <NUM> of the outer tube pump. With this ejection mechanism, the cell sample concentration can be made uniform as described above, and it is possible to prepare start of the liquid feeding.

The cell sample liquid feeding device <NUM> may have a function to feed the sample to a sample introduction unit M3 described later via a liquid feeding tube, besides the above-described functions. For example, the cell sample liquid feeding device <NUM> can suck/feed the sample via a nozzle from a test tube, a well plate, or the like containing the sample, or can also feed the sample by applying pressure to a housing unit that can house the test tube or the like containing the sample.

<FIG> is a schematic conceptual diagram schematically illustrating an exemplary embodiment of the microparticle measurement device <NUM> according to the present technology. The microparticle measurement device <NUM> according to the present technology includes at least the above-described cell sample liquid feeding device <NUM>. Additionally, as necessary, a flow path P, the fluid control unit <NUM>, the light emission unit <NUM>, the light detection unit <NUM>, the analysis unit <NUM>, the sorting unit <NUM>, the electric charging unit <NUM>, recording unit <NUM>, the display unit <NUM>, the input unit <NUM>, the control unit <NUM>, and the like may be provided.

In <FIG>, a liquid feeding tube C1 capable of feeding liquid from the cell sample liquid feeding device <NUM>, a sheath liquid feeding tube C2 capable of feeding liquid from a sheath liquid feeding unit <NUM>, and a liquid drain tube C3 capable of draining liquid to a liquid drain unit <NUM> can be detached as necessary, and these members are disposable. Note that, in the present technology, a part or all of the liquid feeding tube C1 may be similar to the above-described outer tube <NUM>. Furthermore, a microparticle measurement chip M described later is also disposable as necessary.

Each of the units will be described in detail below.

The flow path P may be provided in advance in the microparticle measurement device <NUM> according to the present technology. However, a commercially-available flow path P, a chip provided with the flow path P, or the like can be installed in the microparticle measurement device <NUM> to execute analysis and sorting.

A form of the flow path P that can be used in the microparticle measurement device <NUM> according to the present technology is not particularly limited and can be freely designed. In the present technology, it is particularly preferable to use a flow path P formed inside a substrate T of a two-dimensional or three-dimensional plastic, glass, or the like as illustrated in the microparticle measurement device <NUM> of <FIG>.

Additionally, a flow path width, a flow path depth, a flow path cross-sectional shape, and the like of the flow path P are also not particularly limited and can be freely designed as far as a laminar flow can be formed. For example, a micro flow path having a flow path width of <NUM> or less can also be used in the microparticle measurement device <NUM> according to the present technology. Particularly, a micro flow path having a flow path width of about <NUM> or more and about <NUM> or less can be preferably used in the microparticle measurement device <NUM> according to the present technology.

<FIG> is a schematic view illustrating an exemplary configuration of the microparticle measurement chip M that can be used in the microparticle measurement device <NUM> of <FIG>, and <FIG> is a schematic view illustrating an exemplary configuration of an orifice M1 of the microparticle measurement chip M that can be used in the microparticle measurement device <NUM> of <FIG>. A of <FIG> illustrates a schematic top view, and B of <FIG> illustrates a schematic cross-sectional view corresponding to a cross-section P-P of A. Also, A in <FIG> illustrates a top view, B of <FIG> is a cross-sectional view, and C of <FIG> is a front view. Note that B of <FIG> corresponds to the cross-section P-P in A of <FIG>.

The microparticle measurement chip M is formed by bonding substrate layers Ma and Mb where a sample flow path M2 is formed. The sample flow path M2 can be formed on the substrate layers Ma and Mb by performing injection molding with a thermoplastic resin by using a metal mold. As the thermoplastic resin, it is possible to adopt plastic conventionally known as a material of a microparticle measurement chip, such as polycarbonate, polymethylmethacrylate resin (PMMA), cyclic polyolefin, polyethylene, polystyrene, polypropylene, or polydimethylsiloxane (PDMS).

Additionally, in the microparticle measurement chip M, the sample introduction unit M3 to introduce the sample containing microparticles, a sheath introduction unit M4 to introduce the sheath liquid, and the sample flow path M2 in which a sample flow is introduced and joined with the sheath liquid are formed. The sheath liquid introduced from the sheath introduction unit M4 is fed in two separate directions, and then joined with the sample liquid in a manner interposing the sample liquid between the two directions at a joined portion with the sample liquid introduced from the sample introduction unit M3. Consequently, a three-dimensional laminar flow in which the sample liquid laminar flow is positioned in a middle of sheath liquid laminar flows is formed at the joined portion.

Reference sign M5 illustrated in A of <FIG> represents a suction flow path in order to eliminate clogging or bubbles by temporarily reversing a flow by applying negative pressure to the inside of the sample flow path M2 in the event of the clogging or the bubbles in the sample flow path M2. The suction flow path M5 has one end formed with a suction open portion M51 connected to a negative pressure source such as a vacuum pump. Additionally, the suction flow path M5 has the other end connected to the sample flow path M2 at a communication port M52.

A laminar flow width of the three-dimensional laminar flow is narrowed at narrowed portions M61 (see <FIG>) and M62 (see A and B of <FIG>) each formed such that the area of a vertical cross-section relative to a liquid feeding direction becomes gradually reduced from an upstream side to a downstream side in the liquid feeding direction. After that, the three-dimensional laminar flow is drained as a fluid stream from the orifice M1 provided at one end of the flow path.

The fluid stream jetted from the orifice M1 is made into droplets by applying vibration to the orifice M by a vibration element 105a described below. The orifice M1 is opened in a direction to end surfaces of the substrate layers Ma and Mb, and a cut-away portion M11 is provided between the opened position of the orifice and the end surfaces of the substrate layers. The cut-away portion M11 is formed by cutting the substrate layers Ma and Mb between the opened position of the orifice M1 and the substrate end surfaces such that a diameter L1 of the cut-away portion M11 becomes larger than an opening diameter L2 of the orifice M1 (see C in <FIG>). Preferably, the diameter L1 of the cut-away portion M11 is formed twice larger or more times larger than the opening diameter L2 of the orifice M1 so as not to interfere with movement of droplets discharged from the orifice M1.

The fluid control unit <NUM> includes the sheath liquid feeding unit <NUM> to introduce the sheath liquid to the sheath liquid introduction unit M4. The sheath liquid feeding unit <NUM> includes, for example: a support portion to which a sheath liquid storing unit can be attached: and a sealing portion, and the sheath liquid inside the sheath liquid storing unit is fed to the above-described sheath liquid introduction unit M4 via the sheath liquid feeding tube by pressure to the sealing portion.

The fluid control unit <NUM> may further include the liquid drain unit <NUM>. The liquid drain unit <NUM> collects, for example, clogged matters, bubbles, and the like inside the sample flow path from the suction open portion via the liquid drain tube by a pump function or the like. Additionally, the liquid drain unit <NUM> can also be connected to the sorting unit <NUM> in order to suck a droplet, aerosol, and the like not sorted at the sorting unit <NUM> described below.

Additionally, the fluid control unit <NUM> may include an installation table on which the sheath liquid feeding unit <NUM> and the liquid drain unit <NUM> can be installed. Furthermore, the fluid control unit <NUM> may be formed separately from the microparticle measurement device <NUM> or may be formed as a part of the microparticle measurement device <NUM>.

The light emission unit <NUM> emits light to a microparticle to be analyzed. A kind of light emitted from the light emission unit <NUM> is not particularly limited, but light having a constant light direction, a constant wavelength, and constant light intensity is preferable in order to reliably generate fluorescence and scattered light from a particle. Specifically, for example, a laser, an LED, or the like can be exemplified. In a case of using the laser, a kind thereof is not particularly limited, but it is also possible to use one kind or two or more kinds of combination of: an argon (Ar) ion laser, a helium-neon (He-Ne) laser, a dye laser, a krypton (Cr) laser, a semiconductor laser, a solid laser combining a semiconductor laser with a wavelength conversion optical element, or the like.

The light detection unit <NUM> detects the light generated from the microparticle. The light detection unit <NUM> detects light components of fluorescence, forward scattered light, backscattered light, and the like generated from the microparticle in response to light emission to the microparticle from the light emission unit <NUM>. The components of the fluorescence and necessary scattered light are important light components to obtain optical information (characteristics) of the microparticle P.

As far as light from each microparticle can be detected, a type of the light detection unit <NUM> is not particularly limited, and a known photodetector can be freely selected and adopted. For example, one type or two or more types of following measurement instruments can be freely adopted in combination: a fluorescence measurement instrument, a scattered light measurement instrument, a transmitted light measurement instrument, a reflected light measurement instrument, a diffracted light measurement instrument, an ultraviolet spectrometer, an infrared spectrometer, a Raman spectrometer, a FRET measurement instrument, a FISH measurement instrument, other various spectrum measurement instruments, a so-called multi-channel photodetector in which a plurality of photodetectors is arranged in an array, and the like.

Furthermore, in the present technology, the light detection unit <NUM> preferably has a light receiving element that receives light generated from the microparticle. Examples of the light receiving element can include an area imaging element such as a CCD or a CMOS element, a PMT, a photodiode, and the like.

Furthermore, the light detection unit <NUM> can include a plurality of light receiving elements having different detection wavelength bands. Since the light detection unit <NUM> includes the plurality of light receiving elements having the different detection wavelength bands, intensity of light in a continuous wavelength band can be measured as a fluorescence spectrum. Specifically, for example, it is possible to exemplify: a PMT array or photodiode array in which light receiving elements are arranged one-dimensionally; one in which a plurality of independent detection channels such as two-dimensional light receiving elements like CCDs or CMOSs is arranged; and the like.

The analysis unit <NUM> is connected to the light detection unit <NUM> and analyzes a detection value of light for a microparticle detected by the light detection unit <NUM>.

The analysis unit <NUM> can correct, for example, a detection value of light received from the light detection unit <NUM> and can calculate a feature quantity of each microparticle. Specifically, the feature quantity indicating a size, a form, an internal structure, and the like of the microparticle is calculated from detection values of the received fluorescence, the forward scattered light, and the backscattered light. Additionally, a sorting control signal can also be generated by performing sorting determination on the basis of: the calculated feature quantity; a sorting condition preliminarily received from the input unit; and the like.

The analysis unit <NUM> is not indispensable in the particle measurement device <NUM> according to the present technology, and a state and the like of each microparticle can be analyzed by using an external analysis device or the like on the basis of a detection value of light detected by the light detection unit <NUM>. For example, the analysis unit <NUM> may be implemented by a personal computer or a CPU, and can be made to function by the personal computer or the CPU while further storing a program in a hardware resource including recording media (nonvolatile memory (USB memory or the like), a HDD, a CD, and the like) and the like. Additionally, the analysis unit <NUM> may be connected to each of the units via a network.

The sorting unit <NUM> includes at least: the vibration element 105a that generates a droplet; a deflection plate 105b that changes an electrically-charged droplet in a desired direction; and a collection container that collects droplets. The electric charging unit <NUM> is separately defined in <FIG>, but the electric charging unit is a part of the sorting unit <NUM> and charges electricity on the basis of a sorting control signal generated by the analysis unit <NUM>.

In the microparticle measurement device <NUM> of <FIG>, the vibration element 105a generates a droplet by applying vibration to the orifice M1 as described above. The electric charging unit <NUM> charges positively or negatively the droplet discharged from the orifice M1 of the microparticle measurement chip M on the basis of a sorting control signal generated by the analysis unit <NUM>. Then, an advancing direction of the electrically-charged droplet is changed and sorted in a desired direction by the deflection plate (counter electrode) 105b to which voltage is applied.

Note that the vibration element 105a to be used is not particularly limited and any known vibration element can be freely selected and used. As an example, a piezo vibration element or the like can be exemplified. Additionally, a size of a droplet can be adjusted by adjusting a liquid feeding amount to the flow path P, a diameter of a discharge port, a vibration frequency of the vibration element 105a, and the like, and a droplet including a constant amount of microparticles can be generated.

The memory unit <NUM> stores all of matters relating to measurement, such as a value detected by the light detection unit <NUM>, a feature quantity calculated by the analysis unit <NUM>, a sorting control signal, and a sorting condition input from the input unit.

In the microparticle measurement device <NUM>, the memory unit <NUM> is not indispensable, and an external storage device may be connected. As the memory unit <NUM>, for example, a hard disk or the like can be used. Furthermore, the recording unit <NUM> may be connected to each of the units via the network.

The display unit <NUM> can display all of the matters relating to the measurement such as a value detected by the light detection unit <NUM> and a feature quantity calculated by the analysis unit <NUM>. Preferably, the display unit <NUM> can display, as a scattergram, the feature quantity calculated by the analysis unit <NUM> for each microparticle.

In the microparticle measurement device <NUM>, the display unit <NUM> is not indispensable and an external display device may be connected. As the display unit <NUM>, for example, a display, a printer, or the like can be used.

The input unit <NUM> is a portion operated by a user such as an operator. A user can access the control unit through the input unit <NUM> to control each of the units of the microparticle measurement device <NUM> according to the present technology. Preferably, the input unit <NUM> can set an attention region for the scattergram displayed on the display unit <NUM> and can determine a sorting condition.

In the microparticle measurement device <NUM>, the input unit <NUM> is not indispensable, and an external operating device may be connected. As the input unit <NUM>, for example, a mouse, a keyboard, or the like can also be used.

The control unit <NUM> can control each of the cell sample liquid feeding device <NUM>, the fluid control unit <NUM>, the light emission unit <NUM>, the light detection unit <NUM>, the analysis unit <NUM>, the sorting unit <NUM>, the electric charging unit <NUM>, the recording unit <NUM>, the display unit <NUM>, and input unit <NUM>. The control unit <NUM> may be provided separately for each of the units, and furthermore, may be provided outside the microparticle measurement device <NUM>. For example, the control unit may be implemented by a personal computer or a CPU, and can be made to function by the personal computer or the CPU while further storing a program in a hardware resource including recording media (nonvolatile memory (USB memory and the like), a HDD, a CD, and the like) and the like. Additionally, the control unit <NUM> may be connected to each of the units via the network.

The microparticle measurement device <NUM> according to the present technology can be housed in a biosafety cabinet. Since the microparticle measurement device is housed in the biosafety cabinet, it is possible to prevent: scattering to a surrounding region including a user; and sample contamination. However, the fluid control unit <NUM> is not necessarily housed in the biosafety cabinet and can be connected to the microparticle measurement device <NUM> at an open portion on a wall surface of the biosafety cabinet via each tube at an opened portion.

Claim 1:
A cell sample liquid feeding bag (<NUM>) comprising at least:
an outflow port (<NUM>) from which cell sample liquid flows out;
a bottom portion (<NUM>) at least partly including a slope; and
a first inner tube (<NUM>),
characterized in that
a cross-sectional shape of the bottom portion (<NUM>) has a substantially V-shape;
the bottom portion (<NUM>) includes a reservoir (<NUM>) configured to store cells; and
the first inner tube (<NUM>) extends from the outflow port (<NUM>) toward the reservoir (<NUM>) to a bottom of the V-shape, not contacting the reservoir (<NUM>), and is configured to feed cell sample liquid from a reservoir side toward an outflow port side of the first inner tube (<NUM>) and to whirl up the cells when a liquid is ejected toward the reservoir (<NUM>).