JP6666016B2 - Cell sorting device - Google Patents

Description

  The present invention relates to a cell sorting device.

  Cell materials, unlike other materials, have large individual differences for each cell. Therefore, in cell biology and cell engineering, understanding and manipulating / selecting the individual differences has become increasingly important.

  In recent years, it has become possible to determine the characteristics of individual cells by single-cell analysis technology and to sort the determined cells individually. As a typical single cell analysis technique, a fluorescence-activated cell sorting (FACS) is known.

  Also known is a technique in which a liquid containing labeled cells is flowed through a microchannel using a microchip provided with a microchannel on a substrate such as glass, and the flow is changed according to the characteristics of the cells to perform sorting. ing. For example, Patent Literature 1 proposes a cell sorting apparatus in which a liquid containing labeled cells is caused to flow into a microchannel, and the pressure of the microbubbles is used to push out and sort the selected cells into another flow path. I have.

JP 2013-255441 A

  As described above, since cell materials have large individual differences between cells, it is required to sort rare target cells more reliably and more efficiently.

  Therefore, an object of the present invention is to provide a cell sorting device that can sort target cells more reliably and more efficiently.

  In the cell sorting apparatus according to the present invention, a substrate and a liquid formed on the substrate and including a cell row in which cells are arranged one by one along a flow direction flow from upstream to downstream, and one surface is open. A flow path having a cell sorting area with an open surface, and applying a pressure wave from the opposite side of the open face to the open face to the liquid in the cell sorting area, A first pressure wave applying mechanism that separates from the cell array and moves toward the open surface, and a first pressure wave applying mechanism that is disposed on the open surface side of the cell sorting region and receives a droplet including the first target cell. And one liquid collection tank.

  According to the present invention, the cell sorting device has a cell sorting region having an open surface that is open on one side in a part of a flow path through which a liquid containing cells flows. In the cell sorting region, a first pressure wave applying mechanism for applying a pressure wave to the liquid from the opposite side to the open surface is disposed. By the pressure wave, the target first target cells in the liquid can be moved toward the open surface of the cell sorting area.

  In the cell sorting region, since one surface of the flow channel is partially open, the first target cells that have moved toward the open surface are separated from the liquid flowing through the flow channel as droplets. The droplet containing the first target cell that has left the liquid in this way is collected in the first liquid collection tank arranged on the open side of the cell sorting area.

  In this way, by flowing a liquid containing cells through a flow path having an open surface on one side and applying a pressure wave from the opposite side of the open surface toward the open surface, more reliably and more efficiently. First, the first target cells in the liquid can be sorted.

It is a perspective view explaining the composition of the cell sorting device concerning a 1st embodiment. It is a schematic diagram in the XX section of the cell sorter concerning a 1st embodiment. FIG. 3 is a diagram illustrating a mechanism in which cells in a liquid are separated by irradiation with a femtosecond laser beam. It is a schematic diagram in the longitudinal section along the flow of the cell sorting device of modification (1) of a 1st embodiment. It is a schematic diagram in the longitudinal section along the flow of the cell sorting device of modification (2) of a 1st embodiment. It is a schematic diagram in the longitudinal section along the flow of the cell sorting device of modification (3) of a 1st embodiment. It is the schematic diagram which looked at the cell sorting apparatus which concerns on 2nd Embodiment from the upper side. It is the schematic diagram which looked at the cell sorting apparatus of the modification of 2nd Embodiment from the upper side. It is a schematic diagram in the longitudinal section along the flow of the cell sorting device concerning a 3rd embodiment. It is a schematic diagram in the longitudinal section along the flow of the cell sorting device concerning a 4th embodiment. It is a schematic diagram in the longitudinal section along the flow of the cell sorting device concerning a 6th embodiment. It is a schematic diagram in the longitudinal section along the flow of the cell sorting device concerning an 8th embodiment.

  Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings. Note that the cell sorting devices according to the embodiments can be appropriately combined and configured as long as the effects of the present invention are not impaired.

[First Embodiment]
The overall configuration of the cell sorting device 10 according to the first embodiment of the present invention will be described with reference to FIGS. FIG. 2 shows a configuration of a section taken along line XX in FIG. FIG. 2 is a schematic view for easy understanding, and does not always correspond to an actual scale.

(overall structure)
The cell sorting device 10 includes a substrate 11 on which a channel 12 is formed. The flow channel 12 is provided to penetrate the inside of the substrate 11 from one end 11a to the other end 11b of the substrate 11, and has a cell sorting area 13 having an open surface with an open lower surface. A liquid 14 containing a plurality of cells 15 flows through the channel 12.

  In the present embodiment, the substrate 11 is made of glass of about 20 mm × 50 mm × 1 mm. The flow channel 12 may be formed of a transparent resin such as a polydimethylsiloxane (PDMS) resin, a polymethyl methacrylate (PMMA) resin, and a polyethyl terephthalate (PET) resin. The width of the flow channel 12 can be, for example, about 50 to 500 μm. The depth of the flow channel 12 can be, for example, about 20 to 200 μm. The length of the cell sorting region 13 can be appropriately set within a range sufficiently longer than the size of the cell. For example, when the size of the cells is 10 to 100 μm, the length of the cell sorting region 13 can be 30 to 300 μm or more.

  A first pressure wave applying mechanism 22 is provided on the substrate 11 in the cell sorting area 13, and a first liquid collecting tank 24 is provided below the cell sorting area 13. Although not shown, the first liquid collecting tank 24 is filled with a culture solution.

  The flow path 12 is preferably formed such that the downstream height is substantially equal to the upstream height. Although not shown, it is preferable to include an inflow promotion mechanism that pushes the liquid 14 into the flow channel 12 and a discharge promotion mechanism that draws the liquid 14 out of the flow channel 12. As the inflow promotion mechanism and the discharge promotion mechanism, for example, a syringe pump, a high-performance pressure liquid pump, or the like can be used. When the inflow promotion mechanism and the discharge promotion mechanism are provided, the liquid 14 can flow at a desired speed. It is desirable to adjust the inflow speed of the liquid 14 into the flow channel 12 and the outflow speed of the liquid 14 from the flow channel 12 so that a pressure gradient does not occur in the liquid 14 flowing in the flow channel 12.

  Making the lower surface hydrophilic and the side hydrophobic on the inner surface of the flow channel 12 is also effective for promoting the flow of the liquid 14 from upstream to downstream.

  The liquid 14 flows in the flow channel 12 from the upstream to the downstream as shown by the arrow A. The plurality of cells 15 in the liquid 14 are arranged one by one along the flow direction to form a cell row 16.

  As shown in the XX section of FIG. 2, a first pressure wave applying mechanism 22 (not shown) applies a vertical pressure wave 20 indicated by an arrow B to the liquid 14 in the cell sorting area 13. In the present embodiment, since the open surface of the flow channel 12 is provided on the lower surface, the direction from the opposite side of the open surface to the open surface is a vertical direction from top to bottom. Examples of the pressure wave include a shock wave caused by laser light irradiation and a surface acoustic wave (SAW). Although the substrate 11 is not shown in FIG. 2, the flow channel 12 is provided inside the substrate 11 as shown in FIG.

  In the present embodiment, a pulse laser oscillator that oscillates a femtosecond laser beam 22A is used as the first pressure wave applying mechanism 22, and the femtosecond laser beam 22A is applied to the liquid 14 in the cell sorting region 13 via the objective lens 23. Is irradiated. The femtosecond laser beam 22A can be applied, for example, at a wavelength of 800 nm, a pulse width of about 50 to 300 fs, and a pulse energy of about 10 to 1500 nJ / pulse.

  Below the cell sorting area 13, a first liquid collecting tank 24 for receiving a droplet containing the first target cell 15a is arranged. The first liquid collecting tank 24 can be made of, for example, glass, PDMS resin, PMMA resin, PET resin, or the like. The first liquid collection tank 24 only needs to be able to receive the droplet containing the first target cell 15a, and the size and shape are not particularly limited.

(Action and effect)
In the cell sorting device 10, the femtosecond laser light 22 </ b> A is focused and irradiated on the liquid 14 containing the cells 15 from the pulse laser oscillator as the first pressure wave applying mechanism 22. When the femtosecond laser light 22A is focused and irradiated, cavitation bubbles CB are generated in the liquid 14 by multiphoton absorption as shown in FIG. The cavitation bubble CB is a source of a pressure wave, and a pressure wave (shock wave) PW is induced in the liquid 14. Thereby, the target first target cell 15a moves. The first target cell 15a is separated from the cell row 16 and moves to the open lower surface as shown in FIG.

  In the present embodiment, the first target cells 15a are separated from the cell row 16 by applying a vertical pressure wave 20 from top to bottom to the liquid 14 flowing in the flow channel 12. To the lower side of the liquid 14. Since the lower surface of the flow channel 12 is open, gravity acts when the first target cell 15 a is pushed out of the liquid 14. As a result, the first target cells 15a leave the liquid 14 as droplets and are stored in the first liquid collection tank 24 provided below. Since the first collection tank 24 is filled with the culture solution, the sorted first target cells 15a can be reliably collected.

  As described above, in the present embodiment, since the gravity acts, the first target cells 15a are more reliably and efficiently fractionated as compared with a case where no gravity acts, for example, such as pushing laterally. be able to. Moreover, the sorting of the first target cells 15a in the present embodiment can be performed more easily than in the case where gravity does not act.

  In particular, when a laser that oscillates femtosecond laser light is used, smaller bubbles can be generated with smaller energy than when other laser light is used. Generally, a nanosecond laser beam is irradiated with a pulse width of about 5 to 20 ns and a pulse energy of about 100 to 1000 μJ / pulse. The pulse width of the femtosecond laser light is about one hundred thousandth of the nanosecond laser light, and the pulse energy of the femtosecond laser light is about one thousandth of the nanosecond laser.

  The diameter of the bubble generated by the irradiation of the nanosecond laser beam is set to a minimum of about 100 μm. On the other hand, when a femtosecond laser beam is irradiated, minute cavitation bubbles CB having a diameter of 10 μm or less can be generated. In addition, the lifetime (maintenance time) of the bubble generated by the irradiation of the femtosecond laser beam is 1 μs or less. Since this lifetime is as short as 1/10 of the case of a nanosecond laser beam (10 μs), rapid switching can be expected by using a femtosecond laser beam.

  When a femtosecond laser beam is used, a relationship such as Rmax / Tc ≧ 10 (m / s) is established between the maximum radius Rmax (μm) of the bubble and the maintenance time Tc (μs). This indicates that when the diameter of the cells 15 is 10 μm, a maximum of 1 million cells can be processed per second.

  In a conventional single cell analysis technique such as FACS, the number of cells that can be processed per second (throughput) is about 100,000 at the maximum. In comparison with this, it can be seen that the cell sorting apparatus of the present embodiment can sort cells at a much higher throughput than before.

  As described above, by opening a part of the lower surface of the flow channel 12 through which the liquid 14 including the cell array 16 flows and generating the longitudinal pressure wave 20 from the first pressure wave applying mechanism 22, the desired cell is produced. Can be more reliably and efficiently fractionated.

(Modification)
As the first pressure wave applying mechanism 22, a pulse laser oscillator that oscillates a picosecond laser beam can be used. Also in the case of using picosecond laser light, the same effect can be obtained with the same device configuration as in the case of using femtosecond laser light.

  The first pressure wave applying mechanism 22 may be configured to emit a plurality of laser beams (Modification (1)). For example, as shown in FIG. 4, a plurality of femtosecond laser beams 22 </ b> A collectively irradiate the liquid 14. The interference of the pressure waves 20 caused by the plurality of femtosecond laser beams 22A makes it possible to more reliably sort the first target cells 15a. Such a multi-point focusing system can be configured by, for example, combining a λ / 2 wavelength plate, a beam splitter, and a mirror.

  The greater the number of femtosecond laser beams 22A that are simultaneously focused and irradiated on the liquid 14, the more reliably the first target cells 15a are separated, and a higher effect is obtained.

  As the first pressure wave applying mechanism 22, a pulse laser oscillator that oscillates the nanosecond laser light 22B can be used. In this case, as shown in FIGS. 5 and 6, a metal film 28 is provided in the flow channel 12 as a light absorbing target for absorbing a part of the nanosecond laser light 22B. The metal film 28 can be formed using any metal that absorbs a part of the nanosecond laser light 22B. Examples of the metal that can be used here include gold, chromium, and tungsten. Such a metal may be in the form of a wire.

  The metal film 28 can be provided on the liquid 14 in the cell sorting region 13 as shown in the cell sorting device 10A of FIG. Specifically, the metal film 28 is provided by embedding metal inside the substrate 11 that defines the upper surface of the flow channel 12 in the cell sorting region 13 (Modification (2)). In this case, since a part of the nanosecond laser light 22B irradiated from the first pressure wave applying mechanism 22 is focused and irradiated on the metal film 28, even when the nanosecond laser light is used, the femtosecond laser light is used. Large absorption comparable to light can be achieved.

  A laser oscillator that emits nanosecond laser light is smaller and less expensive than a laser oscillator that emits femtosecond laser light. By providing the metal film 28 that absorbs a part of the nanosecond laser light, even when a smaller and less expensive laser oscillator is used, the same effect as when the femtosecond laser light is used can be obtained.

  The metal film 28 can be provided below the liquid 14 in the cell sorting region 13 as shown in the cell sorting device 10B of FIG. Specifically, the metal film 28 is provided by attaching a metal wire to the substrate 11 that defines the lower surface of the flow channel 12 on the upstream side of the cell sorting region 13 (Modification (3)). In this case, the condensing portion of the nanosecond laser beam 22B emitted from the first pressure wave applying mechanism 22 approaches the cell row 16 in the liquid 14, so that the first target cell 15a is reduced with less energy. It becomes possible to sort. As described above, even when the metal film 28 is provided under the liquid 14 in the cell sorting region 13, the same effect as when the femtosecond laser beam is used can be obtained.

  When a pulse laser oscillator that oscillates microsecond laser light instead of nanosecond laser light is used as the first pressure wave applying mechanism 22, the same effect can be obtained with the same device configuration.

  In the cell sorting region 13, the upper surface of the flow channel 12 may be open. In this case, the upper surface of the flow channel 12 is opened, and the first liquid collecting tank 24 is arranged on the cell sorting region 13, and the first pressure wave is placed on the lower surface side of the substrate 11 in the cell sorting region 13. Except for arranging the application mechanism 22, the configuration can be substantially the same as that of the cell sorting apparatus 10 of FIG. The size of the opening of the first liquid collecting tank 24 is less than about 1 mm, and the first liquid collecting tank 24 stores the culture solution as described above. Even if the opening of the first liquid collecting tank 24 is downward, the contents do not fall because of the surface tension.

  In the case of such a configuration, the pressure wave 20 in the vertical direction acts on the liquid 14 flowing in the flow channel 12 from bottom to top. As described above, the first target cell 15 a is separated from the cell row 16 by the action of the vertical pressure wave 20 and moves to the upper side of the liquid 14. The first target cells 15a are stored in the first liquid collection tank 24 as liquid droplets away from the upper surface of the liquid 14.

  When the open surface is provided on the upper surface of the flow channel 12, the direction from the opposite side of the open surface to the open surface is a vertical direction from bottom to top. Such a configuration can be achieved, for example, by rotating the cell sorting device 10 shown in FIG.

  The direction from the opposite side of the open surface to the open surface is not limited to the vertical direction. By rotating the cell sorting device 10 shown in FIG. 1 at an arbitrary angle about the flow path 12, an arbitrary direction, for example, a lateral direction is set to a direction from the opposite side of the open surface to the open surface. It is also possible. This is applied to the following embodiments.

[Second embodiment]
(overall structure)
Next, the entire configuration of the cell sorting apparatus 10C according to the second embodiment of the present invention will be described with reference to FIG. explain.

  FIG. 7 schematically shows a configuration in which a part of the cell sorting region 13 of the cell sorting device 10C is viewed from the upper side. The arranged first pressure wave applying mechanism 22 is also shown.

  The cell sorting device 10 </ b> C has an interference reduction region 33 including the interference reduction element 32. The interference reduction region 33 can be provided by being buried in the substrate 11 defining the side surface of the flow channel 12 on the upstream side of the portion where the vertical pressure wave is applied from the first pressure wave applying mechanism 22. In the present embodiment, as the interference reducing element 32, a surface acoustic wave device that generates a surface acoustic wave as a pressure wave, particularly, a piezoelectric element is used.

(Action and effect)
The liquid 14 in the cell sorting device 10C flows in the flow channel 12 from upstream to downstream as indicated by the arrow A. The liquid 14 passes through the interference reduction area 33 before being given a longitudinal pressure wave from the first pressure wave applying mechanism 22.

  In the interference reduction area 33, the interference reduction element 32 gives the liquid 14 a lateral pressure wave indicated by an arrow C <b> 1 from one side surface 12 a of the flow channel 12. Due to the action of the lateral pressure wave, the arrangement of the cell rows in the liquid 14 is disturbed, and the cells move toward the opposite side surface 12b of the flow channel 12 as shown by the arrow C2. Thus, the interference between cells is reduced, and the target first target cell 15a is isolated from the preceding and following cells 15b, 15c.

  It is preferable to move the cells to the opposite side surface 12b at a speed equal to or higher than the speed at which the liquid 14 flows in the flow path 12 in the direction of arrow A. In this case, interference between cells in the liquid 14 can be substantially avoided.

  As described above, the first pressure wave applying mechanism 22 applies a vertical pressure wave to the liquid 14 to move the first target cell 15a downward (in the drawing, in the depth direction of the drawing). Since the first target cell 15a is isolated from the preceding and succeeding cells 15b and 15c and interference between the cells is reduced, it is possible to make the longitudinal pressure wave more reliably act on the first target cell 15a. Become. The possibility that the longitudinal pressure wave from the first pressure wave applying mechanism 22 acts on the cells 15b and 15c before and after the pressure wave is reduced, which leads to a reduction in the failure probability of sorting.

  Since interference between cells is reduced, even when a cavitation bubble longer than the length between the cells and having a long operation time (lifetime) is applied, the first target cells 15a can be more reliably sorted. It becomes possible. For example, when the liquid 14 containing cells having a diameter of 10 μm flows at 10 m / s, the first target cells can be collected even when cavitation bubbles having a diameter of about 5 to 10 μm are applied. Cavitation bubbles having a diameter of about 10 μm can be generated, for example, by irradiating femtosecond laser light with a pulse width of about 50 to 300 fs and a pulse energy of about 50 to 1500 nJ / pulse.

(Modification)
The interference reducing element 32 can be provided at any location as long as the pressure wave in the horizontal direction can be applied to the liquid 14 before the pressure wave in the vertical direction is applied from the first pressure wave applying mechanism 22. The interference reducing element 32 may be arranged on any surface of the substrate 11.

  As the interference reducing element 32 that applies a lateral pressure wave to the liquid 14 in the interference reducing region 33, a laser oscillator can be used in addition to the surface acoustic wave device. The laser oscillator serving as the interference reducing element 32 may be the same as or different from the laser oscillator serving as the first pressure wave applying mechanism 22 that applies a longitudinal pressure wave to the liquid 14.

  The interference reducing element 32 can be provided on the side surface 12b of the flow path 12 facing the opposite side. In this case, the cells 15 can be moved from the opposed surface 12b of the flow channel to the one surface 12a in a direction opposite to the above-described example. The interference reducing element 32 may be provided on both sides of the flow path 12. In this case, it is possible to move the cells 15 to the side surface on an arbitrary side of the flow channel 12.

  As shown in the cell sorting device 10D of FIG. 8, the length d of the interference reduction region 33 in the flow direction may be limited by providing the pressure wave absorbing unit 34. As the pressure wave absorber 34, any member that absorbs a part of the lateral pressure wave applied from the interference reducing element 32 can be used, and is not particularly limited. The pressure wave absorbing unit 34 can be configured by arranging, for example, glass, PDMS resin, PMMA resin, and PET resin on the substrate 11.

  The above-described lateral pressure wave from the interference reducing element 32 is similarly applied to a case where an open surface is provided on the upper surface of the flow channel 12 and a vertical pressure wave is applied from bottom to top. Can be applied.

  In the case where the direction from the opposite side of the open surface to the open surface is the horizontal direction, the direction of the pressure wave from the interference reducing element 32 is the vertical direction. In other words, the direction of the pressure wave from the interference reducing element 32 is a direction crossing the direction from the opposite side of the open surface to the open surface.

[Third embodiment]
Next, the entire configuration of the cell sorting apparatus 10E according to the third embodiment of the present invention will be described with reference to FIG. explain.

  In the cell sorting device 10E, the surface acoustic wave device 38 is arranged on the flow channel 12. The surface acoustic wave device 38 in the illustrated cell sorting apparatus 10E is provided on the substrate 11 that defines the upper surface of the flow channel 12, but a part or all of the surface acoustic wave device 38 It may be embedded in the substrate 11 defining the upper surface.

  The surface acoustic wave device 38 generates a ripple (surface tension wave) 36 by making the lower surface of the liquid 14 in the cell sorting region 13 ruffled. In the cell sorting region 13, as described above, the first target cell 15 a moves to the lower side of the liquid 14 by the action of the longitudinal pressure wave 20 from the first pressure wave applying mechanism 22. At this time, the ripples 36 on the surface of the liquid 14 help the droplet including the first target cell 15a to separate from the liquid 14. As a result, the first target cells 15a can be sorted more reliably, which leads to a reduction in the sorting failure probability.

[Fourth embodiment]
Next, the entire configuration of the cell sorting apparatus 10F according to the fourth embodiment of the present invention is described with reference to FIG. explain.

  In the cell sorting device 10F, a separation assisting element 40 is arranged upstream of the cell sorting region 13. Although the auxiliary separation element 40 in the illustrated cell sorting apparatus 10F is provided on the substrate 11 that defines the upper surface of the flow channel 12, a part or all of the auxiliary separation device 40 defines the upper surface of the flow channel 12. Embedded in the substrate 11 to be formed.

  As the separation assisting element 40, a surface acoustic wave device can be used. The surface acoustic wave device applies a longitudinal pressure wave to the liquid 14 to guide the cells 15 in the liquid 14 to the lower surface. As described above, the first target cell 15a moves to the lower side of the liquid 14 by the action of the longitudinal pressure wave 20 from the first pressure wave applying mechanism 22. At this time, since the pressure wave in the vertical direction is applied, it is possible to assist the liquid droplet including the first target cell 15 a to separate from the liquid 14. As a result, the first target cells 15a can be sorted more reliably, which leads to a reduction in the sorting failure probability.

  The cells 15 may be guided toward the lower surface of the liquid 14 by the action of dielectrophoresis. In this case, an electrode for generating a gradient of an electric field is disposed as a separation assisting element 40 on the upstream side of the cell sorting region 13. Since the cells 15 are also guided to the lower surface of the liquid 14 by dielectrophoresis, the first target cells 15a can be sorted more reliably.

[Fifth Embodiment]
In the present embodiment, two surface acoustic wave devices arranged on both sides of the flow path are used as the first pressure wave applying mechanism. The surface acoustic wave device can be embedded in a substrate that defines both sides of the flow path. A standing wave is generated by a surface acoustic wave as a pressure wave generated from two surface acoustic wave devices on both side surfaces of the flow path, and therefore, as in the case where a laser oscillator is used as a first pressure wave applying mechanism. In addition, the first target cells can be more reliably and efficiently sorted.

[Sixth embodiment]
The overall configuration of the cell sorting device 10G according to the sixth embodiment of the present invention will be described with reference to FIG. 11 in which the same components as those of the cell sorting device 10 according to the first embodiment have the same reference numerals.

  In the cell sorting device 10G, the first liquid collecting tank 24A arranged below the cell sorting region 13 includes a plurality of liquid collecting chambers 26 provided in a matrix in the XY directions. The first liquid collecting tank 24A is movable in the XY directions. Each of the liquid collection chambers 26 of the first liquid collection tank 24A is filled with a culture solution.

  As described above, the first target cell 15 a moves to the lower side of the liquid 14 by the longitudinal pressure wave 20 from the first pressure wave applying mechanism 22. Since the culture solution is filled in the liquid collection chamber 26 in which the first target cells 15a are collected as droplets, the sorted first target cells 15a can be reliably collected.

  In addition, the first target cell 15a can be collected in an arbitrary liquid collecting chamber 26 by moving the first liquid collecting tank 24A having the plural liquid collecting chambers 26 in the XY directions.

  Although not shown, the first pressure collecting tank 24A is moved in the X and Y directions by using the second pressure wave applying mechanism, and the second target cell different from the first target cell 15a is subjected to arbitrary liquid collection. It can also be sorted into the chamber 26.

  By providing such a first liquid collecting tank 24A, in the cell sorting device 10G, it is possible to more reliably and efficiently sort desired target cells.

[Seventh embodiment]
In the present embodiment, a plurality of pressure wave applying mechanisms and a plurality of liquid collecting tanks are provided. Specifically, a second pressure wave applying mechanism is further provided on the cell collection area in addition to the first pressure wave applying mechanism, and the second liquid collecting tank is provided in addition to the first liquid collecting tank. Further provided below the region.

  By controlling the irradiation position and irradiation timing by the pressure wave applying mechanism that applies a vertical pressure wave to the liquid in the cell sorting area, the target cells are separated from the cell row at the target collection tank and opened. To the lower surface.

  By arranging a plurality of pressure wave applying mechanisms and a plurality of collecting tanks, multi-sorting for sorting different target cells can be performed more reliably and efficiently. According to the cell sorting device of the present embodiment, since a branch channel for sorting different target cells is not required, multi-sorting can be easily performed.

  Further, by providing the third and fourth additional pressure wave applying mechanisms and the liquid collecting tank in the cell sorting region, more types of different target cells can be sorted.

[Eighth Embodiment]
Next, the entire configuration of the cell sorting apparatus 10H according to the eighth embodiment of the present invention will be described with reference to FIG. explain.

  In the cell sorting device 10H, a collection channel 44 that receives a droplet containing the first target cell 15a is arranged below the cell sorting region 13. A receiving opening 48 is provided in the liquid collection flow path 44 so as to correspond to the cell sorting area 13 of the flow path 12. In the liquid collecting flow path 44, a fluid 46 having a lower speed than the liquid flowing in the flow path 12 flows.

  As described above, the first target cell 15 a moves to the lower side of the liquid 14 by the longitudinal pressure wave 20 from the first pressure wave applying mechanism 22. The droplet containing the first target cell 15a is collected from the cell sorting region 13 of the flow channel 12 into the low-speed fluid 46 via the receiving opening 48 of the collection flow channel 44.

  Providing such a liquid collecting flow path 44 enables the cell sorting apparatus 10H to sort desired cells more reliably and efficiently continuously.

10, 10A, 10B, 10C, 10D, 10E, 10F, 10G, 10H Cell sorting device 11 Substrate 11a One end 11b Other end 12 Flow channel 13 Cell sorting region 14 Liquid 15 cells 15a First target cell 16 Cell row 20 Pressure wave 22 First pressure wave applying mechanism 22A Femtosecond laser light 22B Nanosecond laser light 23 Objective lens 24, 24A First liquid collecting tank 26 Liquid collecting chamber 28 Metal film 32 Interference reduction element 33 Interference reduction area 34 Pressure wave Absorbing part 38 Surface acoustic wave device 40 Separation auxiliary element 44 Liquid collecting channel 46 Fluid 48 Reception opening CB Cavitation bubble PW Pressure wave

Claims (16)

Board and
A flow path formed on the substrate and including a cell row in which cells are arranged one by one along a flow direction flows from upstream to downstream, and has a cell sorting region having an open surface that is open on one side. When,
Applying a pressure wave toward the open surface from the opposite side of the open surface to the liquid in the cell sorting area, separating a first target cell from the cell row and moving toward the open surface. 1 pressure wave applying mechanism,
A first liquid collecting tank disposed on the open surface side of the cell sorting area and receiving a droplet containing the first target cell ;
An inlet promotion mechanism to push the liquid to the flow channel, further comprising cell sorting apparatus according to claim Rukoto the outflow promotion mechanism to draw the liquid into the passage outside.   The first pressure wave applying mechanism is a pulsed laser oscillator that is arranged on the opposite side of the flow path from the open surface and induces a bubble serving as a source of the pressure wave in the liquid. The cell sorting device according to claim 1.   The cell sorter according to claim 2, wherein the open surface is formed by opening a lower surface of the flow channel.   The cell sorting apparatus according to claim 2, wherein the pulse laser oscillator emits femtosecond laser light or picosecond laser light. Board and
A flow path formed on the substrate and including a cell row in which cells are arranged one by one along a flow direction flows from upstream to downstream, and has a cell sorting region having an open surface that is open on one side. When,
Applying a pressure wave toward the open surface from the opposite side of the open surface to the liquid in the cell sorting area, separating a first target cell from the cell row and moving toward the open surface. 1 pressure wave applying mechanism,
A first liquid collection tank disposed on the open surface side of the cell sorting area and receiving a droplet containing the first target cell;
With
The first pressure wave imparting mechanism is a pulsed laser oscillator that is arranged on the opposite side of the open surface of the flow path and that induces a bubble serving as a source of the pressure wave in the liquid,
The pulse laser oscillator oscillates nanosecond laser light or microsecond laser light, and a metal film that absorbs a part of the nanosecond laser light or the microsecond laser light is provided in the flow path. and to that fine胞分winding device. Board and
A flow path formed on the substrate and including a cell row in which cells are arranged one by one along a flow direction flows from upstream to downstream, and has a cell sorting region having an open surface that is open on one side. When,
Applying a pressure wave toward the open surface from the opposite side of the open surface to the liquid in the cell sorting area, separating a first target cell from the cell row and moving toward the open surface. 1 pressure wave applying mechanism,
A first liquid collection tank disposed on the open surface side of the cell sorting area and receiving a droplet containing the first target cell;
With
The first pressure wave imparting mechanism is a pulsed laser oscillator that is arranged on the opposite side of the open surface of the flow path and that induces a bubble serving as a source of the pressure wave in the liquid,
Applying a pressure wave to the liquid from one side surface of the flow path, the interference reduction area having an interference reduction element that disturbs the arrangement of the cells in the cell row and moves toward the opposite side surface of the flow path, opposite from preparative胞分fine you, characterized in that the open surface pressure waves towards is provided upstream from the portion to be imparted device open faces.   The cell sorting apparatus according to claim 6, wherein the interference reducing element is a surface acoustic wave device or a laser oscillator.   The cell sorting apparatus according to claim 6, wherein the length of the interference reduction region in the flow direction is less than 1 mm. Board and
A flow path formed on the substrate and having a cell sorting region having an open surface having an open surface, wherein a liquid including a cell row in which cells are arranged one by one along a flow direction flows from upstream to downstream. When,
Applying a pressure wave toward the open surface from the opposite side of the open surface to the liquid in the cell sorting area, separating a first target cell from the cell row and moving toward the open surface. 1 pressure wave applying mechanism,
A first collection tank disposed on the open surface side of the cell sorting area and receiving a droplet containing the first target cell;
With
A surface acoustic wave device for rippling the surface of the liquid exposed to the cell sorting region is provided on a side of the flow channel opposite to the open surface of the cell sorting region. that fine胞分winding device. Board and
A flow path formed on the substrate and including a cell row in which cells are arranged one by one along a flow direction flows from upstream to downstream, and has a cell sorting region having an open surface that is open on one side. When,
Applying a pressure wave toward the open surface from the opposite side of the open surface to the liquid in the cell sorting area, separating a first target cell from the cell row and moving toward the open surface. 1 pressure wave applying mechanism,
A first liquid collection tank disposed on the open surface side of the cell sorting area and receiving a droplet containing the first target cell;
With
Auxiliary separating element for directing said open surface of said cell region preparative said cell fraction is collected胞分fine you characterized in that provided on the upstream side of the cell sorting region unit. Board and
A flow path formed on the substrate and including a cell row in which cells are arranged one by one along a flow direction flows from upstream to downstream, and has a cell sorting region having an open surface that is open on one side. When,
Applying a pressure wave toward the open surface from the opposite side of the open surface to the liquid in the cell sorting area, separating a first target cell from the cell row and moving toward the open surface. 1 pressure wave applying mechanism,
A first liquid collection tank disposed on the open surface side of the cell sorting area and receiving a droplet containing the first target cell;
With
Said first pressure wave imparting mechanism is arranged on both sides across the flow path, the cells characterized in that it is composed of two surface acoustic wave device for generating a standing wave by the pressure wave Sorting device.   The cell sorting device according to any one of claims 1 to 11, wherein the first liquid collection tank contains a culture solution.   The said 1st liquid collection tank is provided with several liquid collection chambers provided in the XY direction in the shape of a matrix, and is movable in an XY direction, The characterized by the above-mentioned. Cell sorting device. Board and
A flow path formed on the substrate and including a cell row in which cells are arranged one by one along a flow direction flows from upstream to downstream, and has a cell sorting region having an open surface that is open on one side. When,
Applying a pressure wave toward the open surface from the opposite side of the open surface to the liquid in the cell sorting area, separating a first target cell from the cell row and moving toward the open surface. 1 pressure wave applying mechanism,
A first liquid collection tank disposed on the open surface side of the cell sorting area and receiving a droplet containing the first target cell;
With
A pressure wave directed from the opposite side of the open surface to the open surface is applied to the liquid in the cell sorting area, and a second target cell different from a first target cell is separated from the cell row to separate the target cells. A second pressure wave imparting mechanism for moving toward the open surface;
Wherein disposed on the open face side of the cell sorting region, fine胞分winding device you anda second Osamueki tank to receive the liquid droplets containing a second target cell. Board and
A liquid formed on the substrate and including a cell row in which cells are arranged one by one along a flow direction flows from an upstream to a downstream at a first speed, and a cell component having an open surface that is open on one side. A flow path having a taking area;
A pressure wave applying mechanism that applies a pressure wave toward the open surface from the opposite side of the open surface to the liquid in the cell sorting area, and separates target cells from the cell row and moves toward the open surface. When,
A liquid collection channel that is disposed on the open surface side of the cell sorting region, through which a fluid having a second speed smaller than the first speed flows, and that receives a droplet containing the target cell ;
An inlet promotion mechanism to push the liquid to the flow channel, further comprising cell sorting apparatus according to claim Rukoto the outflow promotion mechanism to draw the liquid into the passage outside.   The cell sorting apparatus according to any one of claims 1 to 15, wherein the flow path has a downstream height substantially equal to the upstream height.

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