Cell recovery device and cell recovery method

Abstract

To easily and efficiently collect a target cell captured on a filter while still alive by simple aspiration.SOLUTION: A cell recovery device 10 includes a filter 16 having a cross-sectional arc shape with a hole 16A for capturing a target cell 20 and a capturing wall surface 16B of the hole 16A expanding in diameter toward the cell inflow side, a cylindrical holder 18 for holding the filter 16, a filter unit 12 for immersing the target cell 20 in a recovery solution 26, and a bottom cover 14 that is arranged opposite the back side of the capturing wall surface 16B of the filter 16 after the target cell 20 is captured by the filter 16, forms the bottom of the holder 18, and is placed under the filter unit 12.SELECTED DRAWING: Figure 3

Description

[Technical field]

[0001] The present invention relates to a cell recovery device and a cell recovery method. [Background technology]

[0002] A cell culture method and culture device have been disclosed in which target cells are detached from a filter and cultured (see Patent Document 1). Ultrasonic vibration, application of an electric field, and application of pressure are described as means for detaching cells from the filter.

[0003] It has also been disclosed that a drug solution containing cells is sucked into a pipette from a culture vessel (see Patent Document 2).

[0004] Furthermore, a 3D Pd·Ni filter used for separating circulating tumor cells or rare cells has been disclosed (see Patent Document 3). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] JP 2018-174710 A [Patent Document 2] JP 2021-185749 A [Patent Document 3] Patent No. 5961889 Summary of the Invention [Problem to be solved by the invention]

[0006] However, in the conventional example described in Patent Document 1, it was difficult to increase the recovery rate of the target cells captured by the filter. In the conventional example described in Patent Document 2, it is disclosed that a drug solution containing cells is aspirated into a pipette, but it is not described that the target cells are captured by a filter. When cells are captured using the filter described in Patent Document 3, it is considered that the cells get stuck in the inlet corner of the hole of the filter. In this case, even if the cells are aspirated together with the recovery solution, the cells are difficult to separate from the filter by the suction force alone, so it is considered difficult to increase the recovery rate of the cells.

[0007] An object of the present invention is to simply and efficiently recover target cells captured on a filter while keeping them alive, by simply performing a suction operation. [Means for solving the problem]

[0008] The cell recovery device of the first aspect comprises a filter having a hole in a single component for capturing target cells, the capture wall surface of the hole having an arcuate cross section that expands in diameter toward the cell inflow side, a cylindrical holder for holding the filter, and a bottom cover that is positioned opposite the back side of the capture wall surface of the filter after the target cells have been captured by the filter and that forms the bottom of the holder.

[0009] In this cell recovery device, target cells are captured by a filter held in a holder. A bottom cover constituting the bottom of the holder is disposed opposite the rear side of the capture wall surface of the filter, so that the target cells captured by the filter can be collected by sucking them together with the recovery solution from the capture wall surface side by injecting a recovery solution so that the filter is immersed. In the filter, the capture wall surface of the hole that captures the target cells has an arc-shaped cross section that expands in diameter toward the cell inflow side, so that the target cells are less likely to become trapped on the cell inflow side of the hole compared to when there is a corner on the inflow side of the hole. This makes it easier for the captured target cells to leave the hole.

[0010] In a second aspect, in the cell recovery device according to the first aspect, a recess communicating with the hole is formed on the rear side of the capturing wall surface of the filter.

[0011] In this cell recovery device, a recess is formed on the back side of the capture wall of the filter that is connected to the hole for capturing the target cells, so that the recovery solution easily enters the hole from the recess and passes through to the capture wall side, and therefore the captured target cells are easily separated from the hole when being recovered.

[0012] In a third aspect, in the cell recovery device according to the first aspect, the electroformed metal material of the filter is Ni-Pd.

[0013] In this cell recovery device, the electroformed metal material of the filter is Ni-Pd, and since toxic nickel is not eluted, it is less cytotoxic and easy to recover live cells. Moreover, it is possible to improve the corrosion resistance of the filter while maintaining mechanical properties such as tensile strength.

[0014] A cell recovery method according to a fourth aspect includes the steps of: capturing target cells using a filter unit having a filter with an arc-shaped cross section attached to a cylindrical holder, the filter having a hole in a single component for capturing the target cells, the capture wall surface of the hole expanding in diameter toward the cell inflow side; placing the filter unit on a bottom cover with the bottom cover facing the back side of the capture wall surface of the filter; injecting a recovery solution into the filter unit to immerse the target cells in the recovery solution; and using an aspirator to aspirate and recover the target cells together with the recovery solution from the capture wall surface side.

[0015] In this cell recovery method, target cells are captured using a filter unit. The capture wall surface of the hole that captures the target cells has an arc-shaped cross section that expands in diameter toward the cell inflow side. Next, the filter unit is placed on top of the bottom cover so that the bottom cover faces the back side of the capture wall surface of the filter. Next, a recovery solution is injected into the filter unit to immerse the target cells in the recovery solution. Then, the target cells are sucked in and recovered together with the recovery solution from the capture wall surface side by an aspirator.

[0016] In the filter, the capture wall surface of the hole that captures the target cell has an arc-shaped cross section that expands in diameter toward the cell inflow side, so the target cell is less likely to become trapped on the cell inflow side of the hole compared to when there is a corner on the inflow side of the hole. Therefore, the captured target cell is more likely to leave the hole.

[0017] In a fifth aspect, in the cell recovery method according to the fourth aspect, a recess communicating with the hole is formed on the rear side of the capture wall surface of the filter.

[0018] In this cell recovery method, a recess is formed on the back side of the capture wall of the filter, which is connected to the hole for capturing the target cell, so that the recovery solution can easily pass from the recess into the hole and pass through to the capture wall side. Therefore, when the captured target cell is recovered, the target cell is more likely to leave the hole.

[0019] In a sixth aspect, in the cell recovery method according to the fourth aspect, the recovery solution is pressurized from the rear side of the capture wall surface.

[0020] In this cell recovery method, the recovery solution is pressurized from the back side of the capture wall, causing the recovery solution to flow through the holes toward the capture wall, making it easier for the target cells captured in the holes to separate from the holes.

[0021] In a seventh aspect, in the cell recovery method according to the fourth aspect, the electroformed metal material of the filter is Ni-Pd.

[0022] In this cell recovery method, the electroformed metal material of the filter is Ni-Pd, and since toxic nickel is not eluted, it is less cytotoxic and easy to recover live cells. Moreover, it is possible to improve the corrosion resistance of the filter while maintaining mechanical properties such as tensile strength. Effect of the Invention

[0023] According to the present invention, the target cells captured on the filter can be easily and efficiently recovered while remaining viable by a simple suction operation. [Brief description of the drawings]

[0024] [Figure 1] 3 is a cross-sectional view showing a state in which a target cell is captured by a filter (filter unit) held by a holder in the cell recovery device according to the present embodiment. FIG. [Diagram 2] 10(A) and 10(B) are enlarged cross-sectional views showing the state in which target cells are captured in the holes of the filter. [Diagram 3] FIG. 4 is a cross-sectional view showing a state in which the filter unit is placed on the bottom cover. [Figure 4] FIG. 2 is a cross-sectional view showing a state in which a recovery solution is injected into the filter unit and target cells are immersed in the recovery solution. [Diagram 5] FIG. 11 is a cross-sectional view showing a state in which the recovered solution is pressurized from the rear side of the capture wall surface. [Figure 6] FIG. 11 is a cross-sectional view showing a state in which target cells are being aspirated and collected together with a collection solution from the capture wall surface side by an aspirator. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] Hereinafter, an embodiment of the present invention will be described with reference to the drawings. Components indicated by the same reference numerals in each drawing are the same or similar components. Note that duplicated descriptions and reference numerals in the embodiments described below may be omitted. In addition, all drawings used in the following description are schematic, and the dimensional relationship of each element, the ratio of each element, etc. shown in the drawings do not necessarily match the actual ones. In addition, the dimensional relationship of each element, the ratio of each element, etc. between multiple drawings do not necessarily match.

[0026] [Cell collection device] 1 and 3, a cell collection device 10 according to this embodiment includes a filter unit 12 and a bottom cover 14. The filter unit 12 is configured by attaching a filter 16 to a holder 18.

[0027] In FIG. 2(A), filter 16 is made of, for example, electroformed metal, and has holes 16A for capturing target cells 20 in one member. Capture wall surface 16B of hole 16A has an arc-shaped cross section that expands in diameter toward the cell inflow side. A recess 16C communicating with hole 16A is formed on the back side of capture wall surface 16B in filter 16. Note that recess 16C does not necessarily have to be formed (FIG. 2(B)). Filter 16 in FIG. 2(B) can be obtained by scraping off the portion of recess 16C in FIG. 2(A).

[0028] The electroformed metal material of the filter 16 is, for example, a Ni-Pd (nickel-palladium) alloy. The composition of this alloy is 80% Pd and 20% Ni. The minimum diameter of the holes 16A of the filter 16 (diameter on the cell outflow side) is, for example, 5 to 20 μm.

[0029] The holder 18 is a member that holds the filter 16, and is configured in a cylindrical shape. Specifically, the holder 18 is configured to include an outer member 22 and an inner member 24 that is fitted inside the outer member 22. The centers of the outer member 22 and the inner member 24 penetrate each other in the axial direction. The outer edge of the filter 16 is sandwiched between the outer member 22 and the inner member 24, whereby the filter 16 is held in the holder 18. A screw mechanism (not shown) or the like may be used to continuously generate a sandwiching force between the outer member 22 and the inner member 24.

[0030] When the object to be filtered, such as a body fluid, is passed through the filter unit 12 in the direction of arrow A, the filter 16 allows most of the body fluid cells, such as blood cells, to pass through (in the direction of arrow B), and is able to capture relatively large target cells 20, such as circulating tumor cells (CTCs) and rare cells.

[0031] 3, bottom cover 14 is a member that is disposed opposite the rear side of capture wall surface 16B of filter 16 after target cells 20 are captured by filter 16, and that constitutes the bottom of holder 18. Specifically, bottom cover 14 is a lid that can close, for example, an opening on the cell outflow side of holder 18 (an opening of outer member 22).

[0032] The bottom cover 14 may be configured as a container capable of storing the recovery solution 26, the filter unit 12 (holder 18) may be placed in the container, and the recovery solution 26 may be poured into the container to immerse the target cells 20 captured by the filter 16 in the recovery solution 26. The recovery solution 26 may be, for example, physiological saline (PBS), culture medium, solution, or cell banker.

[0033] [Cell collection method]

[0034] In FIG. 1 to FIG. 6, the cell recovery method according to the present embodiment includes: a step of capturing the target cell 20 using a filter unit 12 in which a filter 16 having an arc-shaped cross section, the filter 16 having a hole 16A for capturing the target cell 20 and a capturing wall surface 16B of the hole 16A expanding in diameter toward the cell inflow side, is attached to a cylindrical holder 18; a step of placing the filter unit 12 on the bottom cover 14 by facing the bottom cover 14 to the rear side of the capturing wall surface 16B of the filter 16; A step of injecting a recovery solution 26 into the filter unit 12 to immerse the target cells 20 in the recovery solution 26; a step of aspirating and recovering the target cells 20 together with the recovery solution 26 from the capture wall surface 16B side by the aspirator 28; has.

[0035] A syringe, a pipette, or the like can be used as the aspirator 28. After the target cells 20 are immersed in the recovery solution 26, the recovery solution 26 may be pressurized from the rear side of the capture wall surface 16B.

[0036] (action) This embodiment is configured as described above, and its operation will be described below. In Fig. 1 to Fig. 6, cells can be collected using the cell collection device 10 according to this embodiment. First, in Fig. 1 and Fig. 2, a target cell 20 is captured from a body fluid using a filter unit 12. A capture wall surface 16B of a hole 16A for capturing the target cell 20 has an arc-shaped cross section that expands in diameter toward the cell inflow side.

[0037] Next, as shown in FIG. 3, the filter unit 12 is placed on the bottom cover 14 so that the bottom cover 14 faces the rear side of the capturing wall surface 16B of the filter 16.

[0038] 4, a recovery solution 26 is poured into the filter unit 12 to immerse the target cells 20 in the recovery solution 26. Since the recovery solution 26 passes through the filter 16, the space between the bottom cover 14 and the filter 16 is filled with the recovery solution 26.

[0039] At this stage, as shown in Fig. 5, the recovery solution 26 may be pressurized from the rear side of the capture wall surface 16B in the direction of the arrow C. Any pressurizing means may be used. For example, a path for applying pressure to the recovery solution 26 on the rear side of the capture wall surface 16B and a pressure applying means such as a syringe may be used, or the filter unit 12 may be pushed down from a slightly raised state.

[0040] Then, as shown in FIG. 6, the target cells 20 are sucked and collected together with the collection solution 26 by the aspirator 28 from the side of the capture wall surface 16B.

[0041] In this embodiment, in the filter 16, the capture wall surface 16B of the hole 16A that captures the target cell 20 has an arc-shaped cross section that expands in diameter toward the cell inflow side, so that the target cell 20 is less likely to get stuck in the cell inflow side of the hole 16A compared to when there is a corner on the inflow side of the hole 16A. Therefore, the captured target cell 20 is more likely to leave the hole 16A.

[0042] In addition, a recess 16C communicating with the hole 16A for capturing the target cell 20 is formed on the back side of the capture wall surface 16B of the filter 16, so that the recovery solution 26 easily enters the hole 16A from the recess 16C and passes through to the capture wall surface 16B side. Therefore, when the captured target cell 20 is recovered, the target cell 20 is more likely to separate from the hole 16A.

[0043] Furthermore, when the recovery solution 26 is pressurized from the rear side of the capture wall surface 16B, the recovery solution 26 flows toward the capture wall surface 16B through the hole 16A. This makes it even easier for the target cells 20 captured in the hole 16A to separate from the hole 16A.

[0044] In addition, the electroformed metal material of the filter 16 is Ni-Pd, which does not elute toxic nickel, so that the cell damage is low and cells can be easily collected alive. Moreover, the corrosion resistance of the filter 16 can be improved while maintaining mechanical properties such as tensile strength.

[0045] As described above, according to this embodiment, the target cells 20 captured on the filter 16 can be easily and efficiently collected while still alive by a simple suction operation.

[0046] (Test Example 1) A test was carried out to confirm the recovery efficiency of the target cells using the filter shown in Figure 2(A) under the following procedures and conditions.

[0047] Set the filter unit in the filtration device, and push 5 mL of PBS-EDTA out from the top of the filtration device using a 10 mL syringe. Keep the filtration device filled with PBS-EDTA without letting air in.

[0048] 1. Spiked samples Blood 2mL + PBS-EDTA 2mL + H1975 (lung cancer cell line) 300 pieces

[0049] 2. Filtration Place this spiked sample in a 15 mL tube, mix gently, and then transfer the entire volume into a 10 mL syringe. The filter unit is connected to a syringe containing blood and set on a syringe pump, and the spiked sample is passed through the filter at 50 mL / h.

[0050] 3. Washing The blood syringe was discarded, and a 10 mL syringe containing 10 mL of PBS-EDTA was connected to the filtration device, which was then set on a syringe pump to pass the buffer through the filter at 50 mL / h.

[0051] 4. Dyeing Prepare 2 mL of antibody solution (dilute the following antibodies in 2 mL of PBS-EDTA). Hoechst33342 2μL (x1 / 1000) EpCAM-PE 10μL (x1 / 200) CD45-FITC. 40μL (x1 / 50) This solution was placed in a 10 mL syringe, attached to a syringe pump, and passed through the filter at 50 mL / h (30 min, left standing at room temperature away from light).

[0052] 5. Washing Discard the antibody syringe and set 10 mL of PBS-EDTA in the syringe pump and pass it through the filter at 50 mL / h.

[0053] 6. Recovery Remove the filter unit from the filtration device and place it on the bottom cover. Place this on a 30 mm dish and pipette 150 μL of medium (10% FBS-RPMI1640) onto the filter (Gilson D200 tip, tip outer diameter 0.9 mm), collect it, and place it in a 1.5 mL tube. This is concentrated by centrifugation, placed in one well of a 96-well plate, and scanned with an inverted microscope. Count Hoechst(+), Hoechst(+)EpCAM(+)CD45(-) cells. 7. Filter after collection After collection, the filters are scanned under a stereomicroscope. Count Hoechst(+), Hoechst(+)EpCAM(+)CD45(-) cells.

[0054] The results are shown in Table 1. According to the results, it was demonstrated that the target cells could be recovered with an average recovery rate of 97.3% simply by aspirating the target cells together with PBS (physiological saline).

[0055] [Table 1]

[0056] (Test Example 2) A test was carried out to confirm the recovery efficiency of target cells using the filter 16 shown in Fig. 2(B). This filter is the same as that shown in Fig. 2(A) except that the S recess 16C is omitted.

[0057] The recovery rate by pipetting immediately after capture was confirmed by a spike test. Items not specifically explained are the same as those in Test Example 1.

[0058] 1. Spiked samples 2. Filtration 3. Washing

[0059] 4. Dyeing 30min,RT

[0060] Prepare 2 mL of antibody solution (dilute the following antibodies in 2 mL of PBS-EDTA). Hoechst33342 x1 / 1000 EpCAM-PE x1 / 100 CD45-FITC. x1 / 50

[0061] 5. Washing 6. Recovery Recover the medium by pipetting 150 μL x 3 times. After concentrating by centrifugation, place in one well of a 96-well plate and scan with an inverted microscope. Count Hoechst(+),Hoechst(+) EpCAM(+) CD45(-) cells.

[0062] 7. Filter after collection After collection, the filters are scanned under a stereomicroscope. Count Hoechst(+),Hoechst(+) EpCAM(+) CD45(-) cells. Antibody used EpCAM-PE, clone 9C4 BioLegend 324206x1 / 100 used CD45-FITC, clone J33 Beckman Coulter A07782x1 / 50 used Hoechst 33342, 10 mg / mL Invitrogen H3570x1 / 1000 used

[0063] The results are shown in Table 2. According to the results, the target cells could be recovered at an average recovery rate of 76.7% simply by aspirating them together with PBS (physiological saline). The average recovery rate of Test Example 1 was 97.3%, which is higher than that of Test Example 2. This is thought to be because the thickness of the filter of Test Example 1 was relatively large due to the presence of recesses, and therefore the shape was stable.

[0064] [Table 2]

[0065] [Other embodiments] An example of an embodiment of the present invention has been described above, but the embodiment of the present invention is not limited to the above, and it goes without saying that various modifications can be made without departing from the spirit and scope of the present invention. [Explanation of symbols]

[0066] 10 Cell collection device 12 Filter unit 14 Bottom cover 16 Filters 16A hole 16B Cell trapping surface 16C Recess 18 Holder 20 target cells 26 Recovery solution 28 Aspirator

Claims

1. a filter unit including a filter having an arc-shaped cross section with holes for capturing target cells, the capture walls of the holes expanding in diameter toward the cell inflow side, and a cylindrical holder for holding the filter, the filter unit immersing the target cells in a recovery solution; a bottom cover that is disposed opposite to the rear side of the capture wall surface of the filter after the target cells are captured by the filter, constitutes the bottom of the holder, and is disposed under the filter unit; A cell recovery device having the same.

2. capturing the target cells using a filter unit having a filter with an arc-shaped cross section, the filter having a hole for capturing the target cells and a capturing wall surface of the hole expanding in diameter toward the cell inflow side, attached to a cylindrical holder; a step of placing a bottom cover under the filter unit so that the bottom cover faces a rear side of the capturing wall surface of the filter; A step of injecting a recovery solution into the filter unit to immerse the target cells in the recovery solution; a step of aspirating and recovering the target cells together with the recovery solution from the capture wall surface side by an aspirator; A cell recovery method comprising the steps of:

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