WO2019240236A1 - Dispositif de lyse de cellules, procédé de lyse de cellules, et procédé de production d'un liquide contenant des composants cellulaires - Google Patents

Dispositif de lyse de cellules, procédé de lyse de cellules, et procédé de production d'un liquide contenant des composants cellulaires Download PDF

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Publication number
WO2019240236A1
WO2019240236A1 PCT/JP2019/023567 JP2019023567W WO2019240236A1 WO 2019240236 A1 WO2019240236 A1 WO 2019240236A1 JP 2019023567 W JP2019023567 W JP 2019023567W WO 2019240236 A1 WO2019240236 A1 WO 2019240236A1
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Prior art keywords
filter
cell
liquid
region
flow path
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PCT/JP2019/023567
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English (en)
Japanese (ja)
Inventor
得仁 菊原
清太 中村
隆文 塚原
大平 小田切
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日立化成株式会社
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/26—Inoculator or sampler

Definitions

  • the present disclosure relates to a cell lysis device, a cell lysis method, and a method for producing a cell component-containing liquid.
  • Japanese Patent Application Laid-Open No. 2015-198595 discloses a cell capture device that can be used for capturing rare cells such as circulating cancer cells (Circulating Tumor Cell).
  • This cell trapping device has a housing in which a filter is accommodated.
  • the present disclosure has been made in view of the above, and provides a cell lysis device, a cell lysis method, and a method for producing a cell component-containing liquid capable of lysing captured cells and recovering liquid after cell lysis.
  • the purpose is to provide.
  • a cell lysis device includes a cell trapping device including a filter in which a plurality of through holes are formed in the thickness direction, and a test solution including cells to be trapped with respect to the cell trapping device
  • a test solution supply unit for supplying cells, a solution supply unit for supplying a solution for dissolving cells in the test solution to the cell capture device, and cells in the test solution from the cell capture device
  • a lysate recovery unit for recovering a lysate containing the components and a recovery unit for recovering the liquid discharged from the cell capture device, and the cell capture device is a space provided above the filter inside A first region, a second region which is a space provided below the filter, a first channel and a second channel connecting the first region and the outside of the device, a second region and the outside of the device
  • a third flow path connecting between the The lysate supply unit is connected to the first channel, the test solution supply unit and the lysate recovery unit are connected to the second channel, and the recovery unit is connected to the third channel.
  • the lysis solution when lysing the cells captured in the first region, the lysis solution is supplied from the lysis solution supply unit via the first channel and the lysis solution via the second channel. Is recovered in the solution recovery unit. Since each of the first flow path and the second flow path is a flow path connected to the first region of the cell trapping device, the lysis solution does not pass through the second region of the cell trapping device. It is recovered in the liquid recovery unit. Thus, in the cell lysis apparatus described above, it is possible to lyse the captured cells and recover the liquid after cell lysis. In addition, since the lysate after cell lysis is collected in the lysate collection unit without passing through the second region of the cell capture device, the lysate contains components of cells different from the cells to be captured. Can be prevented.
  • the filter has a filter region that can come into contact with the liquid introduced into the cell trapping device, and the total area in which the plurality of through holes are formed in the filter region is 10% or less with respect to the area of the filter region. It can be set as a certain aspect.
  • the above configuration prevents mixing of the solution introduced into the first region and the liquid staying in the second region opposite to the first region. Therefore, it is possible to prevent the lysate collected in the lysate collection unit from containing a cell component different from the cell to be captured.
  • the filter may have a filter region that can come into contact with the liquid introduced into the cell trapping device, and the average opening ratio of the through holes in the filter region may be 0.1% or more and 50% or less. .
  • the above configuration prevents mixing of the solution introduced into the first region and the liquid staying in the second region opposite to the first region. Therefore, it is possible to prevent the lysate collected in the lysate collection unit from containing a cell component different from the cell to be captured.
  • the filter can be a metal filter.
  • the plurality of through holes can be formed with high accuracy by electrocast plating using a photoresist, for example. Therefore, it is possible to appropriately capture cells to be captured.
  • the cell lysis apparatus further includes a processing liquid supply unit that supplies a processing liquid for processing cells in the test liquid to the cell trapping device, and the processing liquid supply unit is connected to the first flow path. It can be set as the mode currently performed.
  • cells in the test solution can be suitably processed using a processing solution such as a cleaning solution.
  • the cell lysis apparatus further includes a pretreatment unit on the upstream side of the cell trapping device, and the pretreatment unit is configured to remove at least one of bubbles and foreign substances from at least the lysis solution out of the liquid supplied to the cell trapping device. be able to.
  • the pretreatment unit may be configured to include a filter having a plurality of through holes formed in the thickness direction.
  • the cell lysis apparatus further includes a control unit that controls the movement of the liquid in the cell trapping device, and a liquid feeding unit for the liquid in the cell trapping device, and the control unit includes a first channel and a lysate supply unit. Control of the opening and closing of valves provided between the second flow path and the test liquid supply section and between the second flow path and the solution recovery section, and the operation of the liquid feeding section It can be set as the mode to do.
  • a cell lysis method is a cell lysis method using the cell lysis device described above, and the test solution from the test solution supply unit is passed through the second flow path of the cell capture device.
  • Supplying the lysate through the capture step of capturing the cells to be captured contained in the test solution on one surface of the filter by introducing the sample into the capture device and passing through the filter, and the first flow path of the cell capture device The lysate from the cell is introduced into the first region of the cell trapping device, the cell to be trapped captured by the filter is lysed by the lysate, and the cell traps via the second flow path of the cell trapping device.
  • a recovery step of recovering the lysate containing the components of the cells to be captured that stay in the first region of the device in the lysate recovery unit.
  • the lysis solution is supplied from the lysis solution supply unit via the first channel in the lysis step, and the lysis solution is supplied to the lysis solution recovery unit via the second channel in the recovery step. Collected. Since both the first channel and the second channel are channels connected to the first region of the cell capture device, the lysate does not pass through the second region of the cell capture device. It is collected in the collection unit. Thus, in the cell lysis method described above, it becomes possible to lyse the captured cells and recover the liquid after cell lysis. In addition, since the lysate after cell lysis is collected in the lysate collection part without passing through the second region of the cell capture device, the lysate should contain a cell component different from the target cell. Can be prevented.
  • the method for producing a cell component-containing liquid supplies a test liquid containing cells to be captured to one side of a filter in which a plurality of through holes are formed in the thickness direction.
  • the cells were captured on one side of the filter by passing at least a part of the test solution to the other side of the filter, and the lysate for lysing the cells was supplied to the one side of the filter and captured on the one side. Lysing the cells, and collecting the cell component-containing liquid obtained by lysis of the cells from one side of the filter.
  • the test solution and the lysate are both supplied to one side of the filter, and the lysate containing the lysed cells, that is, the cell component-containing liquid is supplied from the one side of the filter. to recover.
  • the method for producing a cell component-containing liquid it is possible to lyse the captured cells and recover the liquid after cell lysis. Since the cell component-containing liquid is collected without passing through the other side of the filter, it is possible to prevent the cell component-containing liquid from containing cell components different from the cells to be captured.
  • the filter has a filter region that can come into contact with the liquid introduced to the one surface side, and the average opening ratio of the through holes in the filter region may be 0.1% or more and 50% or less.
  • the filter can be a metal filter.
  • a plurality of through holes can be formed with high accuracy by electrocast plating using a photoresist, for example. Therefore, it is possible to appropriately capture cells to be captured.
  • the method for producing the cell component-containing liquid may further include supplying the treatment liquid from one side of the filter to the other side before supplying the test liquid to the filter.
  • cells in the test solution can be suitably processed using a processing solution such as a cleaning solution.
  • a cell lysis device a cell lysis method, and a method for producing a cell component-containing liquid that can lyse captured cells and recover the liquid after lysis.
  • FIG. 1 is a diagram illustrating the configuration of a cell lysis apparatus according to this embodiment.
  • the cell lysis device is a device that captures cells contained in a test solution by filtering the test solution with a filter.
  • This cell lysis device is a device for recovering the lysate after lysing the cells captured by the filter.
  • the test solution is blood and the cell to be captured is a circulating cancer cell (Circulating Tumor Cell: CTC) will be described, but this is not a limitation.
  • the test solution include blood, lymph and the like.
  • Specific cells to be captured include, for example, rare cells such as CTC and blood cells (red blood cells, white blood cells, platelets, etc.).
  • the cell lysis device 100 is provided with a filter for capturing cells therein, and a soft for supplying a treatment liquid (reagent) to the cell capture device 1.
  • a flow path 3 made of a tube and a flow path 4 made of a soft tube for supplying blood to the cell capture device 1 are provided.
  • a treatment liquid supply unit 5 that supplies a treatment liquid (reagent) to the cell capture device 1
  • a lysis solution supply unit 6 that supplies a lysis solution to the cell capture device 1, Is provided.
  • a pretreatment unit 7 is provided on the flow path 3.
  • a test solution supply unit 8 that supplies a test solution to the cell capture device 1, a lysate collection unit 9 that collects a lysate from the cell capture device 1, Is provided.
  • the processing liquid supplied from the processing liquid supply unit 5 provided upstream of the flow path 3 includes a cleaning liquid for cleaning cells and the like captured by the filter, but is not limited to the cleaning liquid.
  • the processing liquid supply unit 5 stores the processing liquids in different containers and can supply the cell capturing device 1 individually from each container. Thereby, a plurality of types of processing liquids can be used individually.
  • Examples of the lysis solution supplied from the lysis solution supply unit 6 provided upstream of the flow path 3 include a lysis solution that dissolves cells (capture target cells) captured by a filter.
  • the type of lysate is selected based on the type of cells to be captured.
  • a surfactant-based solution is used.
  • the treatment liquid supply unit 5 and the solution supply unit 6 are individually provided with flow paths 31 and 32, respectively.
  • the flow paths 31 and 32 are each connected to the flow path 3.
  • Valves V1, V2 are provided on the flow paths 31, 32, respectively. By controlling the opening and closing of the valves V1 and V2, the liquid supplied to the flow path 3 is selectively supplied.
  • the pretreatment unit 7 on the flow path 3 has a function of removing gas from the liquid flowing through the flow path 3 and removing foreign substances in the liquid.
  • the structure and function of the preprocessing unit 7 will be described later.
  • a test liquid supply unit 8 containing a test liquid is provided upstream of the flow path 4. Further, a lysate recovery unit 9 that recovers the lysate from the cell capture device 1 is provided upstream of the flow path 4.
  • the test solution supply unit 8 and the solution recovery unit 9 are individually provided with channels 41 and 42, respectively.
  • the flow paths 41 and 42 are each connected to the flow path 4.
  • valves V3 and V4 are provided on the flow paths 41 and 42, respectively. By controlling the opening and closing of the valves V3 and V4, the liquid supplied to the flow path 3 is selectively supplied.
  • the cell capture device 1 is configured to supply either the treatment solution, the lysis solution, and the blood, instead of supplying them simultaneously.
  • the supply of the liquid to the cell trapping device 1 is switched by opening and closing the valves V1 to V4. For example, when supplying the test solution to the cell capture device 1, the valve V3 is opened and the other valves V1, V2, and V4 are closed.
  • the valves V1 to V4 pinch valves that block a liquid flow by pressurizing and deforming a soft tube can be used.
  • a pump P liquid feeding unit
  • the liquid is supplied by sucking the target liquid by driving.
  • the pump P has a structure capable of changing the flow rate of the liquid in the flow path by changing the rotation speed.
  • a peristaltic pump peripheral pump
  • a liquid such as a processing liquid or blood flows in the direction toward the cell trapping device 1 through the flow path 3 or the flow path 4 and is supplied to the cell trapping device 1.
  • the liquid that has passed through the cell trapping device 1 flows into the recovery unit 11 via the flow path 10.
  • the recovery unit 11 is a container that recovers the liquid flowing from the cell trapping device 1 through the flow path 10.
  • Each unit described above is controlled by the control unit 15. Specifically, the valves V1 to V4 and the pump P are driven by an instruction from the control unit 15.
  • the control unit 15 also controls the valve or pump.
  • the control unit 15 is provided with a program input function for inputting a program that enables control such as driving and stopping for each of the above-described units.
  • the control unit 15 is provided with a drive mechanism for operating each device in order as described above according to the input program.
  • the control unit 15 selects a line through which the liquid flows, the control unit 15 instructs each unit to open / close the valve and drive the pump based on the selection result. (Cell capture device)
  • FIG. 2A is a top view of the cell trapping device 1
  • FIG. 2B is a view taken along arrow IIB-IIB in FIG. 2A.
  • the cell trapping device 1 is configured such that a filter 61 having a plurality of through holes 62 is sandwiched between a lid member 51 and a storage member 52.
  • the filter 61 is disposed in a space formed inside the lid member 51 and the storage member 52 when they are combined.
  • the filter 61 is a metal filter, for example.
  • a plurality of through holes 62 are formed in the thickness direction of the filter 61.
  • the length of one side of the lid member 51 in a plan view can be set to 10 mm to 100 mm, further 15 mm to 70 mm, and particularly 20 mm to 30 mm.
  • the thickness may be 2 mm to 20 mm, further 3 mm to 15 mm, and particularly 5 mm to 10 mm.
  • the metal material used for the filter 61 examples include, but are not limited to, gold, silver, copper, aluminum, tungsten, nickel, chromium, and alloys of these metals.
  • the metal may be used alone, or two or more different materials may be used in combination.
  • the metal may be an alloy with another metal or an oxide of the metal to impart functionality. From the viewpoint of price or availability, it is preferable to use nickel, copper, gold, and a metal containing these as a main component, and it is particularly preferable to use a metal containing nickel as a main component.
  • the main component refers to a component occupying 50% by weight or more of the material forming the filter 61.
  • the filter 61 is formed of a material mainly composed of nickel, the surface of nickel may be plated with gold.
  • the filter 61 may be subjected to a surface treatment using a biocompatible polymer or the like.
  • the polymer used for this purpose include 2-methacryloyloxyethyl phosphorylcholine (MPC).
  • the thickness of the filter 61 can be, for example, 3 ⁇ m to 100 ⁇ m, 5 ⁇ m to 50 ⁇ m, or 5 ⁇ m to 30 ⁇ m.
  • the size of the region in which the through hole 62 of the filter is provided can be set to 25 mm 2 to 1000 mm 2 .
  • the size of the region can be set to 25 mm 2 to 225 mm 2, and further can be set to 25 mm 2 to 100 mm 2 .
  • the processing time can be shortened by setting the size of the region to 25 mm 2 or more.
  • the lid member 51 of the cell trapping device 1 includes a flow path 3A (first flow path) connected to the flow path 3 formed of a soft tube and a flow path 4A (second flow path) connected to the flow path 4. ) And are formed.
  • the lid member 51 is provided with a first region 55 formed above the filter 61 so as to communicate with the flow paths 3A and 4A.
  • a second region 56 is provided in the storage member 52 of the cell trapping device 1.
  • the second region 56 is a space through which the liquid that has passed through the through hole 62 of the filter 61 passes.
  • the second region 56 is formed below the filter 61 so that the depth of the central portion is deeper than the peripheral portion.
  • the storage member 52 is provided with a flow channel 10A (third flow channel) that communicates with the second region 56 and is connected to the flow channel 10 and discharges the liquid in the second region 56 to the outside. ing.
  • the through hole 62 provided in the filter 61 sandwiched between the lid member 51 and the storage member 52 has such a size that the cell 60 to be captured cannot pass through.
  • a region facing the first region 55 and the second region 56 that is, a region that can come into contact with the liquid introduced into the cell trapping device 1 is a filter region. 63.
  • the total area in which the plurality of through holes 62 are formed in the filter region 63 is, for example, 50% or less, 30% or less, or 20% or less with respect to the area of the filter region 63. Or 10% or less.
  • the average aperture ratio of the through holes 62 in the filter region 63 may be, for example, 0.1% or more and 50% or less, 1% or more and 30% or less, or 1% or more and 20% or less.
  • the first region 55 and the second region 56 are disposed to face each other with the filter 61 interposed therebetween. Therefore, the area of the filter region 63 corresponds to the area of the region facing the first region 55 or the second region 56.
  • the total area where the through holes 62 are formed is the total area (cross-sectional area) of the through holes 62 arranged in the filter region 63. The area corresponds to a value obtained by subtracting the area of the filter region 63 where the through hole 62 is not formed (the region where the surface of the filter 61 is present) from the area of the filter region 63.
  • the total area in which the plurality of through holes 62 are formed in the filter region 63 is 50% or less, 30% or less, 20% or less, or 10% or less with respect to the area of the filter region 63, so that the cell trapping device 1 can efficiently lyse cells to be captured and recover the lysate. This point will be described later.
  • the attachment positions of the flow path 3, the flow path 4, and the flow path 10 in the cell trapping device 1 are not limited to the positions shown in FIG. That is, the flow path 3 and the flow path 4 do not need to be arranged to face each other. For example, it is good also as a structure by which the two flow paths 3 and 4 are connected in one of the four side surfaces which comprise the cell capture device 1. FIG. Moreover, it can also be set as the structure by which the flow paths 3 and 4 are connected to the upper surface of the cell capture device 1. FIG. Similarly, the attachment position of the flow path 10 can be changed as appropriate. The attachment positions of the flow paths 3 and 4 and the flow path 10 are changed according to the arrangement of the flow paths 3A, 4A, and 9A of the cell trapping device 1. Therefore, the arrangement of the flow paths 3A, 4A, 9A of the cell capture device 1 can be changed as appropriate. Further, the number of flow paths can be changed as appropriate. (Pre-processing section)
  • FIG. 3A is a top view of the pre-processing unit 7, and FIG. 3B is a view taken along the arrow IIIB-IIIB in FIG. 3A.
  • the inside of the pre-processing unit 7 includes a first region 24 and a second region 26 with a foreign matter removing filter 21 interposed therebetween.
  • the upstream channel of the channel 3 is connected to the first region 24 on the upper side of the pretreatment unit 7, and the downstream channel of the channel 3 is the lower part of the pretreatment unit 7. 2 is connected to the area 26.
  • the pretreatment unit 7 is introduced with a liquid supplied from the upstream processing solution supply unit 5 or the solution supply unit 6.
  • the pretreatment unit 7 has a function of removing bubbles and foreign matters in the liquid before the introduced liquid is discharged to the outside.
  • the length of one side of the lid member 51 in plan view can be set to 10 mm to 100 mm, further 15 mm to 70 mm, and particularly 20 mm to 30 mm. be able to.
  • the thickness may be 2 mm to 20 mm, further 3 mm to 15 mm, and particularly 5 mm to 10 mm.
  • the pretreatment unit 7 removes not only bubbles but also foreign matters in the processing liquid by adopting a configuration in which the foreign matter removing filter 21 having a plurality of through holes 22 is sandwiched between the lid member 28 and the storage member 29. Can do.
  • the planar shape of the lid member 28 and the storage member 29 may be rectangular or circular as in the cell trapping device 1, and is not particularly limited.
  • the foreign matter removing filter 21 is disposed in a space formed inside the lid member 28 and the storage member 29 when they are combined.
  • a plurality of through holes 22 are formed in the thickness direction of the foreign matter removing filter 21.
  • the through-hole 22 provided in the foreign matter removing filter 21 may have a size that allows a plurality of types of treatment liquid and solution to pass therethrough. It is preferable that the through hole 22 has a size that allows any kind of liquid to pass through easily, that is, does not apply a load during passage. Since the treatment liquid or the solution flows through the foreign matter removing filter 21, the size of the through hole 22 can be the same as or smaller than the through hole 62 of the filter 61 of the cell trapping device 1. By setting it as such a structure, when a foreign material is mixed in a process liquid or a solution, a foreign material can be reliably removed by the pre-processing part 7 of the front
  • Examples of the metal material used for the foreign matter removing filter 21 include, but are not limited to, gold, silver, copper, aluminum, tungsten, nickel, chromium, and alloys of these metals.
  • the metal may be used alone, or two or more different materials may be used in combination.
  • the metal may be an alloy with another metal or an oxide of the metal to impart functionality. From the viewpoint of price or availability, it is preferable to use nickel, copper, gold, and a metal containing these as a main component, and it is particularly preferable to use a metal containing nickel as a main component.
  • the main component refers to a component occupying 50% by weight or more of the material forming the foreign matter removing filter 21.
  • the foreign matter removing filter 21 is formed of a material mainly composed of nickel, it is preferable that the nickel surface be plated with gold. Since the oxidation of the filter surface can be prevented by the gold plating, the foreign matter removal performance can be maintained high. It is also preferable to coat the filter surface with a polymer that is harmless to cells. Examples of the polymer used for this purpose include 2-methacryloyloxyethyl phosphorylcholine (MPC).
  • MPC 2-methacryloyloxyethyl phosphorylcholine
  • the thickness of the foreign matter removing filter 21 may be, for example, 3 ⁇ m to 100 ⁇ m, 5 ⁇ m to 50 ⁇ m, or 5 ⁇ m to 30 ⁇ m.
  • the filter is easy to handle and suitable for precision processing.
  • the size of the region in which the through hole 22 is provided can be 25 mm 2 to 1500 mm 2 .
  • the size of the region can be 25 mm 2 to 750 mm 2, and can be 25 mm 2 to 225 mm 2 .
  • the processing time can be shortened by setting the size to 25 mm 2 or more.
  • the foreign matter removing filter 21 has a filter region 23 that can come into contact with the introduced liquid.
  • the total area in which the plurality of through holes 22 are formed is, for example, 50% or less, 30% or less, 20% with respect to the area of the filter region 23. Or less than 10%.
  • the average aperture ratio of the through holes 22 in the filter region 23 may be, for example, 0.1% or more and 50% or less, 1% or more and 30% or less, or 1% or more and 20% or less. It may be 1% or more and 10% or less.
  • a flow path 31A connected to the upstream flow path 3 is formed in the lid member 28 of the pretreatment unit 7.
  • the lid member 28 is provided with a first region 24.
  • the first region 24 is a space for guiding the processing liquid to the through hole 22 of the foreign matter removing filter 21.
  • the first area 24 communicates with the flow path 31A and is formed above the foreign matter removing filter 21.
  • a second region 26 is provided in the storage member 29 of the preprocessing unit 7.
  • the second region 26 is a space for discharging the liquid that has passed through the through hole 22 of the foreign matter removing filter 21 to the outside.
  • the second region 26 is formed below the foreign matter removing filter 21 so that the depth of the central portion is deeper than the peripheral portion.
  • a flow path 31B for discharging the liquid in the second region 26 to the outside is provided in the center of the second region 26, a flow path 31B for discharging the liquid in the second region 26 to the outside.
  • the channel 31 ⁇ / b> B communicates with the second region 26 and is connected to the downstream channel 3.
  • the bottom of the second region 26 is gradually recessed from the end toward the center, and a flow path 31B is provided in the center which is the deepest part. Thereby, the treatment liquid is suitably discharged from the flow path 31B.
  • the space formed by the first region 24 and the second region 26 functions as a bubble retention region having a diameter larger than the diameter of the flow path 3 through which the treatment liquid flows.
  • the diameter of the flow path in the entire cell lysis apparatus 100 is basically the bubble retention region (the space formed by the first region 24 and the second region 26) of the pretreatment unit 7 and the cell trapping device 1. It is preferable that the sizes are the same except for the space inside (the space formed by the first region 55 and the second region 56).
  • the diameter of the bubble retention region means the diameter of the cross section with respect to the moving direction of the treatment liquid in the bubble retention region.
  • the cell capturing device 1 shown in FIG. 2 has a configuration in which the flow path 3A, 4A is provided in the lid member 51 on the upstream side and can be connected to two different flow paths.
  • one flow path 31 ⁇ / b> A is provided in the upstream lid member 28.
  • the end of the one side channel for example, the channel 3 ⁇ / b> A
  • the configuration can be changed to the same configuration as that of the pretreatment unit 7 shown in FIG.
  • the pretreatment unit 7 can be manufactured by using the lid member 28 and the storage member 29 having the same shape as the lid member 51 and the storage member 52 of the cell trapping device 1. Thereby, manufacturing cost can be reduced compared with the case where the pre-processing part 7 which has a shape different from the cell capture device 1, for example is manufactured.
  • the pretreatment unit 7 is provided in the cell lysis device 100 such that the lid member 28 is on the upper side of the storage member 29 in the height direction. Therefore, the end portion 301 on the first region 24 side of the flow channel 31A serving as the inlet for the processing liquid into the internal space (the first region 24 and the second region 26) is the second of the flow channel 32A serving as the discharge port. Above the end portion 302 on the region 26 side. As a result, the processing liquid that passes through the bubble retention region composed of the first region 24 and the second region 26 is introduced from the upper inlet to the first region 24, and the lower outlet of the second region 26. Is discharged to the outside.
  • the bubbles dissolved in the liquid are separated and easily retained as bubbles in the bubble retention region, and the effect of removing bubbles from the treatment liquid can be enhanced.
  • the bubbles separated from the processing liquid stay on the upper side of the first region 24.
  • the lower end of the end portion 301 on the inlet side can be higher than the upper end of the end portion 302 on the outlet side.
  • the lower end of the end portion 301 on the introduction port side can be 0.1 mm to 5 mm above the upper end of the end portion 302 on the discharge port side, and 0.1 mm to 2.5 mm above.
  • the lower end of the end portion 301 on the inlet side can be 0.1 mm to 1.5 mm above the upper end of the end portion 302 on the outlet side.
  • the pretreatment unit 7 By providing the pretreatment unit 7 on the flow path 3, even if bubbles are included in the liquid (treatment liquid or solution), bubbles are generated in the first region 24 in the pretreatment unit 7. Since it can be retained, bubbles can be removed from the liquid while moving the first region 24 and the second region 26. Further, since the foreign matter removing filter 21 is provided, at least a part of the foreign matter contained in the liquid (foreign matter that cannot pass through the through-hole 22) can be captured. It can be supplied to the cell capture device 1.
  • the pretreatment unit 7 may be configured integrally with the cell trapping device 1. That is, in the cell lysis device 100, a device that also functions as the pretreatment unit 7, a channel that connects the pretreatment unit 7 and the cell capture device 1, and a function as the cell capture device 1 may be used.
  • the cell lysis device 100 does not necessarily include the treatment liquid supply unit 5.
  • the pretreatment unit 7 can be arranged at an arbitrary position on the upstream side of the cell capturing device 1. In this case, bubbles of various liquids supplied to the cell trapping device 1 can be removed by the pretreatment unit 7.
  • the pretreatment unit 7 may be located between the cell capture device 1 and the lysate supply unit 6. In this case, at least bubbles in the lysate supplied to the cell trapping device 1 from the lysate supply unit 6 can be removed. (Cell lysis method)
  • the target cells are captured on the filter 61 using the cell capturing device 1 (S01: capturing step).
  • the test solution is supplied from the test solution supply unit 8 to the cell capturing device 1 through the flow path 4.
  • the test liquid passes through the through-hole 62 of the filter 61 from the first region 55 side in the cell trapping device 1, It is discharged from the second region 56 side to the external flow path 10.
  • the cells to be captured do not pass through the through hole 62 of the filter 61 and remain on the filter 61 (the surface of the filter 61 on the first region 55 side).
  • the cells that could not pass through the through hole 62 of the filter 61 remain on the filter 61.
  • the test solution only needs to pass through at least the filter 61, and the drive of the pump P may be stopped before all the test solution is discharged to the flow path 10 in order to prevent air from entering the device.
  • processing related to the captured cells is performed (S02: processing step).
  • the processing liquid is supplied from the processing liquid supply unit 5 to the cell trapping device 1 via the flow path 3.
  • the treatment liquid passes through the through hole 62 of the filter 61 from the first region 55 side in the cell trapping device 1, and the second The gas is discharged from the region 56 side to the external flow path 10.
  • the processing liquid is a cleaning liquid
  • the test liquid staying inside the cell trapping device 1 is discharged to the outside together with the processing liquid through this processing step.
  • a desired process is performed by introduce
  • the processing liquid (the processing liquid supplied last) is supplied to the first region 55, the second region 56, and the flow path of the cell trapping device 1.
  • the drive of the pump P is stopped in a state where 3A, 4A, and 9A are satisfied.
  • target cells remaining on the filter 61 of the cell capture device 1 are lysed (S03: lysis step), and the lysed liquid is recovered (S04: recovery step).
  • the lysis solution is supplied from the lysis solution supply unit 6 to the cell capturing device 1 through the flow path 3 under the control of the control unit 15.
  • the lysing solution is introduced into the device by driving the pump P to the extent that the first region 55 of the cell trapping device 1 is filled.
  • the cells staying in the first region 55 are lysed.
  • the solution is kept waiting in the first region 55 for a predetermined time (for example, several minutes to several tens of minutes).
  • the lysate flows into the lysate recovery unit 9 via the flow path 4.
  • Various methods may be used as the control related to the recovery of the dissolved solution by the control unit 15.
  • a method is used in which the solution from the solution supply unit 6 is introduced into the first region 55 using a pump (not shown) with the valves V1 and V3 closed and the valves V2 and V4 opened. Can do.
  • the liquid (dissolved solution after dissolution) in the first region 55 only needs to be moved to the dissolved solution recovery unit 9, either the treatment solution or the dissolved solution can be used for recovering the dissolved solution.
  • a part of the flow path for example, the flow path 3 between the pretreatment unit 7 and the cell trapping device 1 is opened and air is introduced into the first region 55 without flowing the liquid. it can.
  • FIG. 5 shows the moving direction of the solution in the dissolution step (S03) and the recovery step (S04).
  • the lysate introduced from the flow path 3 and the flow path 3 ⁇ / b> A into the first region 55 of the cell trapping device 1 contacts the cell 60 in the first region 55 and lyses the cell. Thereafter, a lysate containing dissolved cell components is discharged to the channel 4 through the channel 4A.
  • the lysing solution does not pass through the second region 56 to the flow path 10 but passes only through the first region 55 side of the cell trapping device 1.
  • the processing step (S02) is performed before the dissolving step (S03). For this reason, before the lysis solution is introduced into the cell capturing device 1, the device is filled with the treatment solution. Therefore, at the stage where the solution is introduced into the first region 55, the processing solution stays in the second region 56. Since the filter 61 having the through hole 62 is provided between the first region 55 and the second region 56, the solution on the first region 55 side and the second region 56 side are provided via the through hole 62. It is considered that a part of the treatment liquid is mixed (replaced). However, since only a part of the liquid around the through hole 62 is replaced, for example, when there are cells remaining in the second region 56, the cells can be prevented from being lysed by the lysis solution.
  • the lysis solution is moved from the flow path 4 on the first region 55 side to the lysis solution collection unit 9, the lysis solution containing the target cell component is prevented from moving in the second region 56. Accordingly, it is possible to prevent the cell component remaining in the second region 56 from being included in the solution recovered by the solution recovery unit 9.
  • the processing step (S02) may not be performed. Thereby, air can be prevented from entering the device. In addition, it is possible to quickly capture cells.
  • the flow path 3A that is the first flow path is used.
  • the solution is supplied from the solution supply unit 6, and the solution is recovered by the solution recovery unit 9 through the channel 4 ⁇ / b> A that is the second channel. Since the flow paths 3A and 4A are both flow paths connected to the first region 55 of the cell trapping device 1, the lysed solution does not pass through the second region 56 of the cell trapping device 1. It is recovered in the solution recovery unit 9.
  • the cell lysis device 100 it becomes possible to lyse the captured cells and collect the liquid after cell lysis.
  • the lysate after cell lysis contains lysed cell components.
  • the liquid or lysate after cell lysis may be referred to as a cell component-containing liquid.
  • the cell component contained in the cell component-containing liquid varies depending on the type of lysate used, the type of captured cells, and the like. Examples of such cell components include intracellular organelles derived from captured cells; constituent components of intracellular organelles; nucleic acids such as DNA and RNA; proteins, peptides; and exosomes.
  • the control unit 15 includes a valve V1 between the flow path 3A and the 5 treatment liquid supply unit, a valve V2 between the flow path 3A and the lysis liquid supply unit 6, the flow path 4A, The valve V3 between the test solution supply section 8 and the valve V4 between the flow path 4A and the solution recovery section 9 are controlled, and the operation of the pump P functioning as a liquid supply section is controlled.
  • the control unit 15 can control the test solution, the processing solution, and the lysis solution to be appropriately supplied to the cell capturing device 1 and discharged to the outside. Become.
  • a series of operations can be executed under the control of the control unit 15.
  • the total area in which the plurality of through holes 62 are formed in the filter region 63 of the filter 61 is 10% or less with respect to the area of the filter region 63.
  • the average aperture ratio of the through holes 62 is 0.1% or more and 50% or less.
  • the filter 61 can be a metal filter.
  • the plurality of through holes 62 can be formed with high accuracy by, for example, electroforming plating using a photoresist. Therefore, it is possible to appropriately capture cells to be captured.
  • the treatment liquid supply unit 5 is connected to the flow path 3 ⁇ / b> A of the cell trapping device 1 together with the lysis solution supply unit 6.
  • process liquids such as a washing
  • a processing unit 7 is provided. As a result, it is possible to supply the cell capture device 1 with the lysate and the treatment liquid from which bubbles and foreign substances have been removed.
  • the pretreatment unit 7 includes a foreign matter removing filter 21 in which a plurality of through holes 22 are formed in the thickness direction. Thereby, it is possible to more reliably perform the removal of bubbles and foreign matters from the solution or the processing solution.
  • the embodiments of the present disclosure have been described.
  • the cell lysis device and the cell lysis method according to the present disclosure are not limited to the above, and various modifications can be made.
  • the configuration in which the pretreatment unit 7 is provided on the flow path 3 has been described, but the configuration in which the pretreatment unit 7 is omitted may be employed.
  • the structure of the channel outside the cell trapping device 1 can be changed as appropriate.
  • the arrangement of the valves V1 to V4, the arrangement of the pump P, and the like can be changed as appropriate.
  • a cell component-containing solution containing components of cells to be captured is obtained. That is, according to another embodiment of the present disclosure, a method for producing a cell component-containing liquid obtained by cell lysis is provided.
  • the method for producing a cell component-containing liquid according to the present disclosure is a method for producing a cell component-containing liquid by the cell lysis device 100, and passes through the flow path 4 of the cell capturing device 1 and the test liquid from the test liquid supply unit 8. Is introduced into the cell trapping device 1 and allowed to pass through the filter 61, so that the trapping process of trapping the target cells contained in the test solution on one surface of the filter 61 and the flow path 3 of the cell trapping device 1 are performed.
  • a lysis step in which the lysis solution from the lysis solution supply unit 6 is introduced into the first region 55 of the cell capture device 1 and the cells to be captured captured by the filter 61 are lysed by the lysis solution, and the cell capture device 1
  • the said manufacturing method contains another process as needed.
  • the method for producing a cell component-containing liquid according to the present disclosure all matters described for the cell lysis method described above can be used as they are, including the effects.
  • the cell lysis method and the method for producing a cell component-containing solution according to the present disclosure are not limited to the cell lysis device according to the present disclosure as long as the cells to be captured can be lysed.
  • a test solution containing cells to be captured is supplied to one side of a filter having a plurality of through holes formed in the thickness direction, and at least a part of the test solution is supplied.
  • Capturing cells on one side of the filter by passing it to the other side of the filter (capturing step), supplying a lysing solution that dissolves the cells to one side of the filter, and trapping the cells captured on the one side Lysing (lysis step), and recovering the cell component-containing liquid obtained by cell lysis from one side of the filter (recovery step).
  • the cell lysis method includes other steps as necessary.
  • a test liquid containing cells to be captured is supplied to one side of the filter in which a plurality of through holes are formed in the thickness direction, and at least a part of the test liquid is passed through the other side of the filter. As a result, cells are trapped on one side of the filter.
  • the filter used in the capturing step is a cell capturing filter, and the material, the diameter of the through hole, the aperture ratio, and the like can be changed depending on the type of cells to be captured.
  • the filter may be a metal filter.
  • the metal used for the filter include gold, silver, copper, aluminum, tungsten, nickel, chromium, and alloys of these metals, and examples include nickel, copper, gold, and metals containing these as main components. Or a metal based on nickel.
  • the main component refers to a component occupying 50% by weight or more of the material forming the filter. Thereby, a plurality of through-holes can be formed with high accuracy by, for example, electroforming plating using a photoresist.
  • the filter By forming a plurality of through holes with high accuracy, it is possible to appropriately capture the cells to be captured.
  • the filter is formed from a material mainly composed of nickel, the surface of nickel may be plated with gold. Gold plating can prevent oxidation of the filter surface. For this reason, the adherence to specific cells (for example, CTC) to be captured becomes uniform, and the reproducibility of data can be improved.
  • the filter may be subjected to a surface treatment using a biocompatible polymer or the like.
  • the thickness of the filter can be, for example, 3 ⁇ m to 100 ⁇ m, 5 ⁇ m to 50 ⁇ m, or 5 ⁇ m to 30 ⁇ m.
  • the size of the region in which the through hole of the filter is provided can be, for example, 25 mm 2 to 1000 mm 2 , can be 25 mm 2 to 225 mm 2, and can be 25 mm 2 to 100 mm 2. .
  • the processing time can be shortened by setting the size of the region to 25 mm 2 or more.
  • the filter is provided with a region that can come into contact with the liquid introduced to one side of the filter, that is, a filter region.
  • the total area where the plurality of through holes are formed is, for example, 50% or less, 30% or less, 20% or less, or 10% with respect to the area of the filter region. It can be as follows.
  • the average aperture ratio of the through holes in the filter region may be 0.1% or more and 50% or less, 1% or more and 30% or less, or 1% or more and 20% or less. % Or more and 10% or less.
  • the filter 61 described above in this specification can be used as the cell trapping filter in the cell lysis method of the present disclosure.
  • test solution from one side (cell capture surface) side of the filter to the other side of the filter (surface opposite to the cell capture surface)
  • mechanical operation such as driving by a pump, centrifugation, or the like
  • a manual operation such as extrusion or suction with a syringe, addition with a pipette, or the like can be used.
  • a lysis solution for lysing cells is supplied to one side of the filter. Thereby, lysis of the cells captured on one side of the filter is performed. Examples of the solution include the solution described above in the present specification.
  • the cell is lysed on one side of the filter where the cells are captured.
  • the cell component-containing liquid obtained by cell lysis is collected from one side of the filter.
  • the cells captured by the filter are lysed by lysis treatment on the filter, and the cell component-containing liquid obtained by lysis stays on one side of the filter without passing through the filter.
  • the recovery step the cell component-containing liquid staying on one side of the filter is recovered from the one side of the filter. For this reason, in the cell lysis method of the present disclosure, the cell component-containing liquid can be collected from the one surface side of the filter without being mixed with the liquid staying on the other surface side of the filter.
  • the cell lysis method and the method for producing a cell component-containing liquid according to the present disclosure include a processing step of supplying a processing solution from one side of the filter to the other side before the capturing step of supplying the test solution to the filter. Can do. Thereby, a desired process can be performed with respect to the cell in a test liquid, for example using process liquids, such as a washing
  • the supplied processing liquid can be removed from one side of the filter.
  • the cell lysis method and the method for producing a cell component-containing solution of the present disclosure can include a treatment step for performing a predetermined treatment on the cells after the capture step and before the lysis step.
  • a predetermined amount of the treatment liquid is supplied to one side of the filter.
  • the supply amount of the treatment liquid can be an amount that fills at least the space region on the other surface side of the filter.
  • the dissolving solution can be added to the processing solution staying on one side of the filter. Further, the treatment liquid staying on one side of the filter may be replaced with a solution. Thereby, even if the cells remain on the other surface side of the filter, the cells can be prevented from being lysed by the lysis solution. Further, by collecting the lysate from one side of the filter, it is possible to prevent the lysate containing the components of the target cells from being collected together with the liquid on the other side of the filter. Therefore, it is possible to prevent the cell components remaining on the other surface side of the filter from being mixed into the lysate collected after the lysis treatment.
  • the cell lysis method or the method for producing a cell component-containing solution of the present disclosure can include a washing step for washing the filter or the like with a washing solution before or after the capturing step. Thereby, the cell used as capture
  • the cells to be captured in the test solution are efficiently captured and lysed, and the cell components derived from the cells to be captured are collected.
  • a cell component-containing solution containing a high concentration can be obtained.
  • the cell component-containing liquid can contain, for example, components of cells to be captured captured at a capture rate of 20% or more, or 30% or more.
  • the cell component-containing liquid can be a liquid concentrated to 1/5 or more, or 1/10 or more, for example, as a volume comparison with the test liquid.

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Abstract

La présente invention concerne un dispositif de lyse de cellules comprenant : un dispositif de capture de cellules (1) disposé au plan interne avec un filtre (61) dans lequel de multiples trous traversants (62) sont formés; une unité d'alimentation de liquide de test (8) qui alimente un liquide de test; une unité d'alimentation de lysat (6) qui alimente un lysat; une unité de récupération de lysat (9) qui récupère un lysat; et une unité de récupération (11) qui récupère un liquide. Le dispositif de capture de cellules (1) comprend au plan interne : une première région (55), qui est un espace disposé au-dessus du filtre (61), une seconde région (56), qui est un espace disposé en-dessous du filtre (61), un premier trajet d'écoulement (3A) et un second trajet d'écoulement (4A), qui relient la première région à l'extérieur du dispositif; et un troisième trajet d'écoulement (10A) qui relie la seconde région à l'extérieur du dispositif. L'unité d'alimentation de liquide de traitement et l'unité d'alimentation de lysat sont reliées au premier trajet d'écoulement, l'unité d'alimentation de liquide de test et l'unité de récupération de lysat sont reliées au second trajet d'écoulement, et l'unité de récupération est reliée au troisième trajet d'écoulement.
PCT/JP2019/023567 2018-06-13 2019-06-13 Dispositif de lyse de cellules, procédé de lyse de cellules, et procédé de production d'un liquide contenant des composants cellulaires WO2019240236A1 (fr)

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KR101959447B1 (ko) * 2012-04-06 2019-03-18 삼성전자주식회사 시료 중의 표적물질을 효율적으로 처리하는 방법
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JP2015192642A (ja) * 2014-03-28 2015-11-05 日立化成株式会社 細胞捕捉装置、前処理部付き細胞捕捉デバイス、及び前処理部
JP2017192394A (ja) * 2014-07-30 2017-10-26 日立化成株式会社 血中希少細胞捕獲方法
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