WO2019239514A1 - Dispositif de lyse cellulaire et procédé de lyse cellulaire - Google Patents

Dispositif de lyse cellulaire et procédé de lyse cellulaire Download PDF

Info

Publication number
WO2019239514A1
WO2019239514A1 PCT/JP2018/022548 JP2018022548W WO2019239514A1 WO 2019239514 A1 WO2019239514 A1 WO 2019239514A1 JP 2018022548 W JP2018022548 W JP 2018022548W WO 2019239514 A1 WO2019239514 A1 WO 2019239514A1
Authority
WO
WIPO (PCT)
Prior art keywords
cell
region
flow path
liquid
filter
Prior art date
Application number
PCT/JP2018/022548
Other languages
English (en)
Japanese (ja)
Inventor
得仁 菊原
清太 中村
隆文 塚原
大平 小田切
Original Assignee
日立化成株式会社
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 日立化成株式会社 filed Critical 日立化成株式会社
Priority to PCT/JP2018/022548 priority Critical patent/WO2019239514A1/fr
Priority to PCT/JP2019/023567 priority patent/WO2019240236A1/fr
Publication of WO2019239514A1 publication Critical patent/WO2019239514A1/fr

Links

Images

Classifications

    • 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 invention relates to a cell lysis apparatus and a cell lysis method.
  • Patent Document 1 discloses a cell trapping device having a housing in which a filter is housed.
  • the present invention has been made in view of the above, and an object of the present invention is to provide a cell lysis apparatus and a cell disruption method capable of lysing captured cells and recovering the liquid after lysis.
  • a cell lysis apparatus includes a cell trapping device including a filter in which a plurality of through holes are formed in a thickness direction, and a target for the cell trapping device.
  • a test liquid supply unit that supplies a test liquid containing cells
  • a processing liquid supply unit that supplies a processing liquid for processing the cells in the test liquid to the cell capture device
  • the cell capture A lysate supply unit for supplying a lysate for lysing cells in the test solution to the device, and a lysate containing the components of the cells in the test solution from the cell capture device are collected.
  • a lysate recovery unit, and a recovery unit that recovers the liquid discharged from the cell capture device wherein the cell capture device includes a first region that is a space provided on the upper side of the filter. Under the filter A second region that is a space provided in the first region, a first channel and a second channel that connect the first region and the outside of the device, and a second channel that connects the second region and the outside of the device.
  • the treatment liquid supply unit and the lysis solution supply unit are connected to the first flow channel, and the test solution supply unit and the lysis solution recovery unit are connected to the second flow channel.
  • the recovery unit is connected to the flow path, and the recovery unit is connected to the third flow path.
  • 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 lysis. In addition, since the lysed solution after lysis is collected in the lysate collection unit without passing through the second region of the cell trapping device, the lysis solution contains a cell component different from the target cell. Can be prevented.
  • the apparatus further includes a control unit that controls movement of the liquid in the cell trapping device, and a liquid feeding unit of the liquid in the cell trapping device, and the control unit includes the first flow path and the treatment liquid.
  • the control unit includes the first flow path and the treatment liquid.
  • the total area in which the plurality of through holes are formed is based on the area of the filter region. It can be set as the aspect which is 10% or less.
  • a cell lysis method is a cell lysis method using the above-described cell lysis apparatus, and the sample to be supplied from the test solution supply unit through the second flow path of the cell capture device.
  • a capturing step of capturing a target cell contained in the test solution by the filter by introducing a test solution into the cell capturing device and passing the filter, and the first flow of the cell capturing device Through the path, by introducing the treatment liquid from the treatment liquid supply unit into the cell capture device, a treatment step for performing treatment with the treatment liquid, and through the first flow path of the cell capture device, A lysing step of introducing the lysate from the lysate supply unit into the first region of the cell trapping device, and lysing the target cells captured by the filter with the lysate; Through said second flow path of the chair, having a recovery step of recovering said solution collecting section lysates containing components of said target cells remaining in the first area of the cell trapping device.
  • 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 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 method described above, it becomes possible to lyse the captured cells and recover the liquid after lysis. In addition, since the lysed solution after lysis is collected in the lysate collection unit without passing through the second region of the cell trapping device, the lysis solution contains a cell component different from the target cell. Can be prevented.
  • a cell lysis apparatus and a cell disruption method capable of lysing captured cells and recovering the liquid after lysis.
  • FIG. 1 is a view for explaining 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.
  • the cell lysis device is a device for recovering the lysate after lysing the cells captured by the filter.
  • the test solution includes blood, lymph, and the like, and the specific cells to be captured include rare cells such as CTC, and cells in blood (red blood cells, white blood cells, platelets, etc.).
  • a cell lysis device 100 includes a cell capture device 1 in which a filter for capturing cells is provided, and a soft tube for supplying a treatment liquid (reagent) to the cell capture device 1. And a flow path 4 made of a soft tube for supplying blood to the cell trapping device 1 is 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. Further, in the case of using a plurality of types of processing liquids, the processing liquid supply unit 5 stores the processing liquids in different containers, and can individually supply the cell trapping device 1 from each container. By doing so, a plurality of types of treatment liquids can be used individually. In the case of using a plurality of types of processing liquids, it is possible to provide a configuration in which an individual flow path is provided for each container and a flow path connected to the flow path 3 among the individual flow paths is switched by a selection valve.
  • 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, and for example, a surfactant-based lysate 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 and V2 are provided on the flow paths 31 and 32, respectively, and the liquid supplied to the flow path 3 is selected and supplied by controlling the opening and closing of the valves V1 and V2.
  • 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, and the liquid supplied to the flow path 3 is selected and supplied by controlling the opening and closing of the valves V3 and V4.
  • the cell capture device 1 is configured to supply any one of the treatment solution, the lysis solution, and the blood instead of supplying them simultaneously. Accordingly, the liquid supply 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. As the valves V1 to V4, pinch valves that block the flow of the soft tube by applying pressure deformation can be used.
  • a pump P liquid feed
  • the liquid is supplied by aspirating the target liquid by driving the unit.
  • 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 (peristaltic pump) that sequentially moves a peristaltic point by pressurization of the soft tube can be used.
  • 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 4 and is supplied to the cell trapping device 1.
  • the liquid that has passed through the cell trapping device 1 has a structure that flows into the recovery unit 11 via the flow path 10.
  • the collection unit 11 is a collection container for the liquid flowing from the cell capture device 1 through the flow path 10.
  • the control of each unit described above is performed by the control 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. In addition to the illustrated valve and pump, when a valve or pump is provided, 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 enabling control of driving, stopping, etc. for each of the above-described units. A drive mechanism that operates in order is added.
  • the control unit 15 selects a line through which the liquid flows, and based on the selection result, the control unit 15 instructs each unit to open and close the valve and drive the pump.
  • 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 made of, for example, metal and has a plurality of through holes 62 formed in the thickness direction.
  • the dimensions of the cell trapping device 1 can be such that the length of one side of the lid member 51 in plan view can be 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 may be used as an alloy with another metal or a metal oxide in order 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 filter 61 is formed of a material mainly composed of nickel, the nickel surface may be plated with gold.
  • the filter 61 may be subjected to a surface treatment using a biocompatible polymer or the like.
  • the thickness of the filter 61 can be 3 ⁇ m to 100 ⁇ 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 size exceeds 1000 mm 2 , dead space increases.
  • it is less than 25 mm 2 , the processing time becomes long.
  • 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 a first region 55 formed above the filter 61 in communication with the flow paths 3A and 4A.
  • the storage member 52 of the cell trapping device 1 is formed below the filter 61 so that the central portion has a depth deeper than the peripheral portion and becomes a space through which the liquid that has passed through the through hole 62 of the filter 61 passes. Two regions 56 are provided. Further, the storage member 52 has a flow path 10A (third flow path) that communicates with the second area 56 and is connected to the flow path 10 to discharge the liquid in the second area 56 to the outside. Provided. The through hole 62 provided in the filter 61 sandwiched between the lid member 51 and the storage member 52 has a size that prevents the cell 60 to be captured from passing through.
  • a region sandwiched between 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.
  • the total area in which the plurality of through holes 62 are formed in the filter region 63 is 10% or less with respect to the area of the filter region 63.
  • the area of the filter region 63 the first region 55 and the second region 56 of the filter 61 are opposed to each other with the filter 61 interposed therebetween as shown in FIG. 2. 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 in which the through holes 62 are formed is the total 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 10% or less with respect to the area of the filter region 63, so that Cell lysis and recovery of the lysate can be performed efficiently. 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. In other words, the flow channel 3 and the flow channel 4 do not have to be arranged to face each other.
  • the two flow channels 3 and 4 are connected to one of the four side surfaces constituting the cell trapping device 1. It is good also as a structure to be. Moreover, it can also be set as the structure by which the flow paths 3 and 4 are connected to an upper surface.
  • the mounting 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, and 9A of the cell capture device 1 can be changed as appropriate. Further, the number of flow paths can be changed as appropriate.
  • 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 preprocessing 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 a lower part of the pretreatment unit 7. 2 is connected to the area 26.
  • Such a pre-processing unit 7 introduces the liquid supplied from the upstream processing liquid supply unit 5 or the solution supply unit 6 and removes bubbles and foreign matters in the liquid until it is discharged to the outside. It has a function.
  • the dimensions of the pretreatment unit 7 can be such that the length of one side of the lid member 51 in a plan view can be 10 mm to 100 mm, further 15 mm to 70 mm, and particularly 20 mm to 30 mm. It can be.
  • 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.
  • the foreign matter removing filter 21 has a plurality of through holes 22 formed in the thickness direction.
  • the size of the through-hole 22 provided in the foreign matter removing filter 21 needs to allow all of a plurality of types of treatment liquid and solution to pass through, but any type of liquid can easily pass through. That is, it is preferable that the size is such that no load is applied during passage.
  • the size of the through hole 22 of the foreign matter removing filter 21 in the pretreatment unit 7 through which the treatment liquid or the lysing solution flows can be the same or smaller than the through hole 62 of the filter 61 of the cell trapping device.
  • the metal material used for the foreign matter removing filter 21 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 may be used as an alloy with another metal or a metal oxide in order 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 foreign matter removing filter 21 is formed of a material mainly composed of nickel, it is preferable that the nickel surface is plated with gold.
  • 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 surface of the filter with a polymer that is harmless to cells. However, in this case, adhesion to cells is not required.
  • the thickness of the foreign matter removing filter 21 is preferably 3 to 100 ⁇ m.
  • the size of the region 24 provided with the through hole 22 of the filter can be set to 25 mm 2 to 1500 mm 2 .
  • the size of the region 24 can be set to 25 mm 2 to 750 mm 2, and further can be set to 25 mm 2 to 225 mm 2 . If the size exceeds 1500 mm 2 , the dead space increases. If the size is less than 25 mm 2 , the processing time becomes long.
  • the lid member 28 of the pretreatment section 7 is formed with a flow path 31A (connected to the upstream flow path 3 and formed above the foreign matter removal filter 21 in communication with the flow path 31A.
  • a first region 24 serving as a space for guiding the liquid to the through hole 22 of the foreign matter removing filter 21 is provided.
  • the storage member 29 of the pretreatment unit 7 is formed below the foreign matter removing filter 21 so that the central portion is deeper than the peripheral portion, and has passed through the through hole 22 of the foreign matter removing filter 21.
  • a second region 26 serving as a space for discharging the liquid to the outside is provided.
  • the storage member 29 has a flow path 31B that communicates with the second area 26 and is connected to the flow path 3 on the downstream side, and discharges the liquid in the second area 26 to the outside.
  • 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 larger diameter than the diameter of the flow path 3 through which the treatment liquid is circulated.
  • the diameter of the flow path in the whole cell lysis apparatus 100 is basically the bubble retention area (the space formed by the first area 24 and the second area 26) of the pretreatment unit 7 and the cell trapping device from the viewpoint of reproducibility. It is preferable that the sizes are the same except for the space inside 1 (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 can be used as the pretreatment unit 7 described above.
  • 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.
  • the upstream lid member 28 is provided with one flow path 31A. Therefore, the end of the cell capture device 1 shown in FIG. 2 on the side connected to the external flow path (flow path 3) of the flow path on one side (for example, the flow path 3A) is closed, and the cell capture device 1 is attached to the lid member 51.
  • 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 above the storage member 29 in the height direction, whereby an internal space (first region 24 and second region 26).
  • 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 upper side is located above the end portion 302 on the second region 26 side of the flow channel 32A serving as the discharge port.
  • 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 internal pressure of the processing liquid or the test liquid that has reached the bubble retention region is lowered at a stroke, and the bubbles dissolved in the liquid are separated and easily retained as bubbles, thereby enhancing the effect of removing bubbles from the processing liquid. it can. Then, the bubbles separated from the processing liquid move and stay above the first region 24.
  • the processing liquid passes through the through hole 22 of the foreign matter removing filter 21
  • foreign matter that cannot pass through the through hole 22 is captured by the foreign matter removing filter 21.
  • the processing liquid from which bubbles and foreign matters have been removed is discharged from the second region 26 to the downstream flow path 32 through the flow path 32A.
  • the positional relationship between the end portion 301 on the first region 24 side of the flow channel 31A serving as the inlet and the end portion 302 on the second region 26 side of the flow channel 32A serving as the discharge port is as follows.
  • the lower end of the discharge port can be higher than the upper end of the end portion 302 on the discharge port 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 is 0.1 mm to 2.5 mm above. be able to.
  • 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 first region in the pretreatment unit 7 can be used even when bubbles are included in the liquid (treatment liquid or solution). Since the air bubbles stay in 24, the air bubbles are removed while moving in 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 having a function as the pretreatment unit 7, a flow path connecting the pretreatment unit 7 and the cell capture device 1, and the cell capture device 1 may be used.
  • Cell lysis method Next, a cell lysis method using the cell lysis device 100 will be described with reference to FIG.
  • 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. It should be noted that the test solution only needs to pass through at least the filter 61, and even if the driving of the pump P is stopped before all the test solution is discharged to the flow path 10 in order to prevent air from entering the device. Good.
  • 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, 2 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 lysis solution is introduced into the device by driving the pump P until the lysis solution fills the first region 55 of the cell trapping device 1.
  • the lysate is introduced into the device by driving the pump P until the lysis solution fills the first region 55 of the cell trapping device 1.
  • the solution staying in the first region 55 is recovered to the solution recovery unit 9 (S04: recovery step).
  • the lysate flows into the lysate recovery unit 9 via the flow path 4.
  • valves V2 and V3 may be closed, the valves V1 and V4 may be opened, and the processing liquid from the processing liquid supply unit 5 may be introduced into the first region 55 using a pump or the like (not shown). Since the liquid in the first region 55 (dissolved solution after dissolution) only needs to be moved to the dissolved solution recovery unit 9, both the processing solution and the dissolved solution can be used. Further, 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 the lysate staying in the first region 55 is introduced by introducing air without flowing the liquid. The solution recovery unit 9 may recover the solution.
  • FIG. 5 shows the moving direction of the solution in the dissolution step (S03) and the recovery step (S04).
  • the lysate introduced into the first region 55 of the cell trapping device 1 from the channel 3 and the channel 3 ⁇ / b> A contacts the cell 60 in the first region 55 and lyses the cell.
  • the lysate containing the 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 lysis step (S03), the lysis solution is filled in the device before the lysis solution is introduced into the cell capture device 1. It becomes a state. 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 interposed through the through hole 62. A part of the treatment liquid is considered to be 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 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.
  • each of the flow paths 3A and 4A is a flow path connected to the first region 55 of the cell trapping device 1, the lysed solution after lysis passes through the second region 56 of the cell trapping device 1. Instead, it is recovered in the solution recovery unit 9.
  • the cell lysis device 100 it becomes possible to lyse the captured cells and recover the liquid after lysis.
  • the lysate recovery unit 9 since the lysed solution after lysis is recovered by the lysate recovery unit 9 without passing through the second region 56 of the cell trapping device 1, the lysate contains components of cells different from the target cells. Can be prevented.
  • 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 is equal to the area of the filter region 63. 10% or less.
  • the first region can be prevented by preventing vibration of the cell trapping device 1 or the like. Mixing of the solution introduced into 55 and the liquid staying in the second region 56 on the opposite side can be suppressed.
  • the pretreatment unit 7 may not be provided.
  • the configuration of the flow path outside the cell trapping device 1 in the cell lysis apparatus 100 can be changed as appropriate. Further, the arrangement of the valves V1 to V4, the arrangement of the pump P, and the like can be changed as appropriate.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Microbiology (AREA)
  • Sustainable Development (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

La présente invention concerne un dispositif de lyse cellulaire qui comprend : un dispositif de capture de cellules (1) pourvu intérieurement d'un filtre (61) dans lequel de multiples trous traversants (62) sont formés ; une unité d'alimentation en liquide de test (8) qui fournit un liquide de test ; une unité d'alimentation en liquide de traitement (5) qui fournit un liquide de traitement ; une unité d'alimentation en lysat (6) qui fournit un lysat ; une unité de récupération de lysat (9) qui récupère le lysat provenant du dispositif de capture de cellules (1) ; et une unité de récupération (11) qui récupère le liquide déchargé. Le dispositif de capture de cellules (1) comprend intérieurement : une première région (55), qui est un espace disposé au-dessus du filtre (61), une seconde région (56), qui est un espace disposé au-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 en liquide de traitement et l'unité d'alimentation en lysat sont reliées au premier trajet d'écoulement, l'unité d'alimentation en 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/JP2018/022548 2018-06-13 2018-06-13 Dispositif de lyse cellulaire et procédé de lyse cellulaire WO2019239514A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
PCT/JP2018/022548 WO2019239514A1 (fr) 2018-06-13 2018-06-13 Dispositif de lyse cellulaire et procédé de lyse cellulaire
PCT/JP2019/023567 WO2019240236A1 (fr) 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

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/JP2018/022548 WO2019239514A1 (fr) 2018-06-13 2018-06-13 Dispositif de lyse cellulaire et procédé de lyse cellulaire

Publications (1)

Publication Number Publication Date
WO2019239514A1 true WO2019239514A1 (fr) 2019-12-19

Family

ID=68841867

Family Applications (2)

Application Number Title Priority Date Filing Date
PCT/JP2018/022548 WO2019239514A1 (fr) 2018-06-13 2018-06-13 Dispositif de lyse cellulaire et procédé de lyse cellulaire
PCT/JP2019/023567 WO2019240236A1 (fr) 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

Family Applications After (1)

Application Number Title Priority Date Filing Date
PCT/JP2019/023567 WO2019240236A1 (fr) 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

Country Status (1)

Country Link
WO (2) WO2019239514A1 (fr)

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007014239A (ja) * 2005-07-06 2007-01-25 Matsushita Electric Ind Co Ltd 微生物検出方法および検出装置
JP2012019710A (ja) * 2010-07-13 2012-02-02 Sysmex Corp 生体物質濃縮装置
US20120309004A1 (en) * 2010-10-29 2012-12-06 Samsung Electronics Co., Ltd. Micro-device and methods for disrupting cells
US20130264205A1 (en) * 2012-04-06 2013-10-10 Samsung Electronics Co., Ltd. Method of processing target material in a sample
JP2014223058A (ja) * 2013-04-22 2014-12-04 日立化成株式会社 がん細胞捕捉装置、処理液キット及び処理液キットの製造方法
JP2015188376A (ja) * 2014-03-28 2015-11-02 日立化成株式会社 処理液キット及び細胞捕捉装置

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6759233B2 (en) * 2000-04-13 2004-07-06 Millipore Corporation Method of plasmid recovery and apparatus for doing so
KR20160137552A (ko) * 2014-03-28 2016-11-30 히타치가세이가부시끼가이샤 세포 포착 장치, 전처리부 부착 세포 포착 디바이스 및 전처리부
WO2016017755A1 (fr) * 2014-07-30 2016-02-04 日立化成株式会社 Procédé de capture de cellules rares dans le sang
US11519916B2 (en) * 2015-06-08 2022-12-06 Arquer Diagnostics Limited Methods for analysing a urine sample

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007014239A (ja) * 2005-07-06 2007-01-25 Matsushita Electric Ind Co Ltd 微生物検出方法および検出装置
JP2012019710A (ja) * 2010-07-13 2012-02-02 Sysmex Corp 生体物質濃縮装置
US20120309004A1 (en) * 2010-10-29 2012-12-06 Samsung Electronics Co., Ltd. Micro-device and methods for disrupting cells
US20130264205A1 (en) * 2012-04-06 2013-10-10 Samsung Electronics Co., Ltd. Method of processing target material in a sample
JP2014223058A (ja) * 2013-04-22 2014-12-04 日立化成株式会社 がん細胞捕捉装置、処理液キット及び処理液キットの製造方法
JP2015188376A (ja) * 2014-03-28 2015-11-02 日立化成株式会社 処理液キット及び細胞捕捉装置

Also Published As

Publication number Publication date
WO2019240236A1 (fr) 2019-12-19

Similar Documents

Publication Publication Date Title
JP6484942B2 (ja) 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
US20220090997A1 (en) Filter Arrangement Using Elution Fluid and Method for Using the Same
JP2009038998A (ja) 細胞分離装置
CN108841713B (zh) 理化处理单个细胞的微流控芯片、微流控装置及使用其理化处理单个细胞的方法
JP6124285B2 (ja) 循環がん細胞捕捉装置
JP2020535840A (ja) 細胞濃縮にクロスフローろ過を利用するためのシステム
JP2016086736A (ja) 血中希少細胞含有液の製造方法
WO2019239514A1 (fr) Dispositif de lyse cellulaire et procédé de lyse cellulaire
JP5512754B2 (ja) 磁場を利用する自己細胞抽出装置およびこれを利用する方法
JP2017181096A (ja) 希少細胞を捕捉する方法
JP6164375B2 (ja) 細胞捕捉デバイス
JP4803064B2 (ja) フィールドフローフラクショネーション装置
CN113846012A (zh) 一种细胞集中分选***及分选方法
KR20190103145A (ko) 마이크로 유체 장치, 마이크로 유체 시스템 및 입자 분리 방법
JP7191389B2 (ja) デジタルマイクロ流体デバイス中の少量の不均一懸濁液のろ過のための方法
JP4762521B2 (ja) バイオセパレーション用キットおよびその使用方法
WO2017130563A1 (fr) Procédé de capture de cellules rares
KR102106960B1 (ko) 시료 여과 장치 및 이를 이용한 여과 방법
JP7020488B2 (ja) 流体デバイス及びその使用
WO2023110319A1 (fr) Dispositif et procédé de séparation d'agglomérats tridimensionnels
CN110831684A (zh) 浓缩装置
CN117500600A (zh) 用于使用针对微流体设备的至少一个电极捕获至少一个有核细胞的方法和装置
JPS6125040A (ja) 微小粒子分析／分離装置におけるサンプル供給方法およびその装置

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 18922330

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 18922330

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: JP