JP2023024283A - Cell recovery method and cell recovery kit - Google Patents

Abstract

To provide a cell recovery method that can efficiently recover a cell.SOLUTION: A cell recovery method includes connecting a tube to a cell culture substrate in which cells are cultured, sucking cells from the cell culture substrate into the tube, and closing one or multiple points of the tube.SELECTED DRAWING: Figure 1

Description

TECHNICAL FIELD The present invention relates to cell technology, and relates to a cell collection method and a cell collection kit.

Embryonic stem cells (ES cells) are stem cells established from early human or mouse embryos. ES cells have pluripotency capable of differentiating into all cells existing in living organisms. Human ES cells are now available for cell transplantation therapy for many diseases such as Parkinson's disease, juvenile diabetes, and leukemia. However, ES cell transplantation also has obstacles. In particular, transplantation of ES cells can provoke immune rejection similar to the rejection that follows unsuccessful organ transplantation. In addition, from an ethical standpoint, there are many criticisms and objections to the use of ES cells established by destroying human embryos.

Against this background, Professor Shinya Yamanaka of Kyoto University has developed induced pluripotent stem cells (iPS) by introducing four genes: OCT3/4, KLF4, c-MYC, and SOX2 into somatic cells. cells) were successfully established. As a result, Professor Yamanaka was awarded the 2012 Nobel Prize in Physiology or Medicine (see Patent Documents 1 and 2, for example). iPS cells are ideal pluripotent cells free from rejection and ethical issues. Therefore, iPS cells are expected to be used for cell transplantation therapy.

Japanese Patent No. 4183742 JP 2014-114997 A

There is a demand for a technique that can efficiently collect not only iPS cells but also various cells. Accordingly, one object of the present invention is to provide a cell collection method and a cell collection kit capable of efficiently collecting cells.

A method for collecting cells according to an aspect of the present invention includes connecting a tube to a cell culture vessel in which cells are cultured, sucking cells from the cell culture vessel into the tube, and closing one or more locations of the tube. including doing and

In the method for collecting cells described above, the cells may be in a cryopreservation solution.

In the above method of collecting cells, the cells may be in a medium or buffer.

The method of collecting cells described above may further comprise counting the number of cells in between the occluded portions of the tube.

The method for collecting cells described above may further include cutting a plurality of blocked portions of the tube to obtain portions of the tube that are blocked at both ends.

The cell harvesting method described above may further comprise counting the number of cells in the portion of the tube.

The above method of harvesting cells further comprising calculating the number of tube sections required based on the number of cells in the tube section, the length of the tube section, and the number of cells required. You can stay.

The above method for collecting cells may further include joining a tube containing cells inside and a tube containing a substance inside, and bringing the substance into contact with the cells.

The method of collecting cells described above may further include placing the tube portion in a container.

The cell collection method described above may further comprise freezing the liquid within the portion of the tube.

In addition, a method for collecting cells according to an aspect of the present invention includes the steps of: sticking a suction needle into a container housing a bag containing cells from the outside of the bag; and sucking the cells through the suction needle. and including.

The above cell collection method may further include sticking an injection needle into the bag from outside the container, and introducing fluid into the bag through the injection needle.

In the method for collecting cells described above, a guide for positioning the suction needle in the container may be used in inserting the suction needle into the bag from the outside of the container.

Further, a cell collection kit according to an aspect of the present invention includes a cell culture vessel, a tube connected to the cell culture vessel, an aspirator for aspirating cells in the cell culture vessel into the tube, and a tube for closing the tube. a tube sealer;

In the cell collection kit described above, the tube may indicate the closed position.

The cell collection kit described above may further comprise a container containing the portion of the closed and cut tube.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide a cell collection method and a cell collection kit capable of efficiently collecting cells.

1 is a schematic perspective view of a cell collection kit according to a first embodiment; FIG. 1 is a schematic perspective view of a cell collection kit according to a first embodiment; FIG. 1 is a schematic cross-sectional view of a cell collection kit according to a first embodiment; FIG. 1 is a schematic perspective view of a cell collection kit according to a first embodiment; FIG. 1 is a schematic perspective view of a cell collection kit according to a first embodiment; FIG. 1 is a schematic perspective view of a cell collection kit according to a first embodiment; FIG. 1 is a schematic perspective view of a cell collection kit according to a first embodiment; FIG. 1 is a schematic top view of a cell collection kit according to a first embodiment; FIG. 1 is a schematic diagram of an aseptic welding apparatus according to a first embodiment; FIG. 1 is a schematic diagram of an aseptic welding apparatus according to a first embodiment; FIG. 1 is a schematic perspective view of a cell collection kit according to a first embodiment; FIG. FIG. 4 is a schematic perspective view of a cell collection kit according to a second embodiment; FIG. 4 is a schematic perspective view of a cell collection kit according to a second embodiment; FIG. 4 is a schematic perspective view of a cell collection kit according to a second embodiment; FIG. 12A is a schematic perspective view and a schematic side view of a cell collection kit according to a third embodiment. FIG. FIG. 10A is a schematic side view and a schematic cross-sectional view of a cell collection kit according to a third embodiment; FIG. 11 is a schematic side view of a cell collection kit according to a third embodiment; FIG. 10A is a schematic side view and a schematic cross-sectional view of a cell collection kit according to a third embodiment; FIG. 10A is a schematic perspective view and a schematic cross-sectional view of a cell collection kit according to a third embodiment. FIG. 12A is a schematic perspective view and a schematic side view of a cell collection kit according to a third embodiment. FIG. FIG. 11 is a schematic side view of a cell collection kit according to a third embodiment; FIG. 21(b) is an enlarged view of the portion surrounded by A in FIG. 21(a).

Embodiments of the present invention are described below. In the following description of the drawings, the same or similar parts are denoted by the same or similar reference numerals. However, the drawings are schematic. Therefore, specific dimensions and the like should be determined by referring to the following description. In addition, it is a matter of course that there are portions with different dimensional relationships and ratios between the drawings.

(First embodiment)
The cell collection kit according to the first embodiment, as shown in FIG. and a tube sealer 80 for closing the tube 20.

At least a portion of cell incubator 10 may be gas permeable. The gas permeable portion may be gas permeable and liquid impermeable. The gas permeable part may be, for example, a filter. Cell incubator 10 need not be gas permeable. The volume of cell incubator 10 may be variable.

As shown in FIGS. 2 and 3, the cell incubator 10 includes, for example, a housing 11 having an opening and a lid 12 for the opening. At least part of the housing 11 may be gas permeable, and at least part of the lid 12 may be gas permeable. 2 and 3 show an example in which the lid 12 is provided with a gas-permeable filter 13. FIG. A gas permeable portion is impermeable to, for example, dust, dirt, bacteria, and viruses.

Cells are cultured within the housing 11 . The size and shape of the housing 11 are not particularly limited as long as cells can be cultured inside. Cell incubator 10 may be a flask. Examples of materials for the housing 11 include resin and glass. The housing 11 may be transparent. In the housing 11, the cells may be adherently cultured, suspended cultured, two-dimensionally cultured, or three-dimensionally cultured.

At least part of the surface constituting the housing 11 may or may not be coated with a coating agent for cell adhesion. Examples of cell adhesion coatings include matrigel, collagen, polylysine, fibronectin, vitronectin, gelatin, and laminin. Alternatively, when cells are cultured in suspension, at least a portion of the surface constituting housing 11 may be coated with a coating agent that suppresses cell adhesion. Examples of coating agents that inhibit cell adhesion include poly(2-hydroxyethyl methacrylate). Moreover, at least part of the surface forming the housing 11 may be hydrophilic. The inside of the housing 11 may be sterilized. Examples of sterilization include autoclave sterilization, irradiation with radiation such as gamma rays, and sterilization with UV irradiation.

Examples of cells cultured in an incubator include somatic cells, but are not particularly limited. Examples of cells include fibroblasts, nerve cells, retinal epithelial cells, hepatocytes, β cells, renal cells, blood cells, dental pulp stem cells, keratinocytes, dermal papilla cells, oral epithelial cells, megakaryocytes, T cells, NK cells, NKT cells, chondrocytes, cardiomyocytes, myocytes, vascular cells, epithelial cells, factor-introduced cells, reprogramming cells, and stem cells, but are not limited thereto. Examples of stem cells include, but are not limited to, mesenchymal stem cells, somatic stem progenitor cells, pluripotent stem cells, ES cells, and iPS cells.

A medium for culturing cells is appropriately selected according to the type of cells. For example, if the cells are somatic cells, a somatic cell culture medium such as differentiated cell culture medium is selected. If the cells are stem cells, a stem cell medium suitable for stem cells is selected. The medium may be a gel, a liquid, or a flowable solid. Flowable solids include agar and temperature sensitive gels.

When the medium is gel-like, the medium may contain a polymer compound. Polymer compounds include, for example, gellan gum, deacylated gellan gum, hyaluronic acid, rhamsan gum, diutan gum, xanthan gum, carrageenan, fucoidan, pectin, pectic acid, pectinic acid, heparan sulfate, heparin, heparitin sulfate, keratosulfate, chondroitin sulfate , deltamansulfate, rhamnansulfate, and salts thereof. In addition, the medium may contain methylcellulose.

Alternatively, the medium may contain poly(glycerol monomethacrylate) (PGMA), poly(2-hydroxypropyl methacrylate) (PHPMA), Poly (N-isopropylacrylamide) (PNIPAM), amine terminated, carboxylic acid terminated, maleimide terminated, N-hydroxysuccinimide (NHS ) ester terminated, triethoxysilane terminated, Poly (N-isopropylacrylamide-co-acrylamide), Poly (N-isopropylacrylamide-co-acrylic acid), Poly (N-isopropylacrylamide-co-butylacrylate), Poly (N-isopropylacrylamide-co-methacrylic acid), Poly (N-isopropylacrylamide-co-methacrylic acid-co-octadecyl acrylate), and N-isopropylacrylamide. In the present disclosure, a gel medium or gel medium includes a polymer medium.

The tube 20 may be connected to the lid 12 of the cell incubator 10, or may be connected to the housing 11 of the cell incubator 10 as shown in FIG. The portion of housing 11 of cell incubator 10 to which tube 20 is connected is not particularly limited. For example, the tube 20 may be connected at or near a corner of the housing 11 of the cell incubator 10 or connected above the cell adhesion surface of the housing 11 of the cell incubator 10 . The tube 20 may be inserted even inside the housing 11 of the cell incubator 10 . The tube 20 may reach the bottom inside the housing 11 of the cell incubator 10 . This can facilitate recovery of the solution near the bottom surface. The tube 20 may reach the corner of the bottom surface inside the housing 11 of the cell incubator 10 or the periphery of the corner. The portion of the lid 12 of the cell incubator 10 to which the tube 20 is connected is also not particularly limited. Tube 20 may be sterilely joined to cell incubator 10 .

Tube 20 is a flexible tube. The size of the tube 20 is not particularly limited. Moreover, the cross-sectional shape of the tube 20 is not particularly limited. The cross-sectional shape of the tube 20 may be circular or polygonal. The tube 20 is made of resin, for example. The resin is, for example, synthetic resin. Examples of synthetic resins include polyvinyl chloride (PVC). The tube 20 is made of, for example, a heat-sealable material. Tube 20 can be closed by tube sealer 80 . Tube 20 may be transparent.

For example, when the tube 20 is made of resin, the tube 20 is closed by sandwiching it with the tube sealer 80 while heating the tube 20 . In addition, the method for closing the tube 20 is not limited to the above, and light processing, laser light processing, friction processing, rubbing processing, heat processing without pressure, and pressure processing without heat may be used. can be done.

The aspirator 30 shown in FIG. 1 includes, for example, a syringe containing a fluid, and a plunger inserted into the syringe and capable of moving within the syringe. The volume may be changeable. Alternatively, the suction device 30 may be a flexible bellows or bag.

Next, a method for collecting cells according to the first embodiment will be described. The method for collecting cells according to the first embodiment includes, as shown in FIG. This includes aspirating cells into tube 20 and occluding one or more portions of tube 20, as shown in FIG.

When cells are adherently cultured in the cell culture vessel 10 shown in FIG. 1, the cells may be detached from the inner wall of the cell culture vessel 10 with a detachment agent. Within cell incubator 10, cells may be suspended in a cryopreservation medium. Alternatively, cells may be suspended in a medium or buffer within the cell incubator 10 .

The tube 20 may be connected to the cell incubator 10 before the cells are cultured in the cell incubator 10, or connected to the cell incubator 10 while the cells are being cultured in the cell incubator 10. Alternatively, the cells may be connected to the cell incubator 10 after being cultured in the cell incubator 10 .

As shown in FIG. 5 , the aspirator 30 is used to aspirate fluid containing cells in the cell culture vessel 10 into the tube 20 . When the fluid inside the cell incubator 10 is sucked by the aspirator 30, the inside of the cell incubator 10 is decompressed, and the gas around the cell incubator 10 flows through the gas-permeable portion of the cell incubator 10. Get inside 10. If the container of cell incubator 10 is variable and cell incubator 10 is not gas permeable, aspirator 30 will aspirate the fluid within cell incubator 10 and the container of cell incubator 10 will contract. It should be noted that not all the cells in the cell incubator 10 may be aspirated. A part of the cells in the cell incubator 10 may be aspirated and the remaining cells may continue to be cultured in the cell incubator 10 . By analyzing some of the aspirated cells, it is possible to verify the cell culture process. Also, when a variable volume container such as a syringe or a bag is connected to the cell culture device 10 and the fluid in the cell culture device 10 is sucked by the aspirator 30, the fluid is supplied from the variable volume container into the cell culture device 10. and the pressure reduction in the cell incubator 10 may be suppressed. In this case, the cell incubator 10 may not be provided with gas permeable portions. Thereby, cells can be collected from the cell culture vessel 10 in a closed system without being exposed to the outside air.

After the fluid containing cells is sucked into the tube 20, a tube sealer 80 is used to close one or more points 21A, 21B, 21C, . . . of the tube 20 as shown in FIG. The location where the tube 20 is blocked is arbitrary. The tube 20 may be closed at regular intervals. The tube 20 may have pre-marked occlusion locations, and one or more locations of the tube 20 may be occluded according to the indicated occlusion locations. The closed position may be printed on the tube 20 . The number of cells in between adjacent occluded portions of tube 20 may be counted.

Each of the closed portions 21A, 21B, 21C, . . . of the tube 20 has a predetermined width. As shown in FIGS. 7 and 8, a plurality of blocked portions 21A, 21B, 21C, . The number of cells in each of the portions 20a, 20b, 20c, 20d of tube 20 closed at both ends may be counted. Based on the length of each section of tube 20, the number of cells counted, and the number of cells required, the number of sections of tube 20 required may be calculated.

A tube closed at both ends containing cells therein and a tube closed at both ends containing a substance inside may be aseptically joined to contact the substance with the cells.

For example, a tube 20a containing cells inside and a tube 45 containing a substance inside shown in FIG.

The aseptic joining device comprises, for example, holders 121, 132 holding tubes 20a and 45 arranged partially side by side, and a cutter 55 movable between the holders 121, 132. FIG. Cutter 55 is heatable. As shown in FIG. 9B, the cutter 55 moves between the holders 121 and 132 and melts and cuts the tubes 20a and 45 respectively. The melted and cut portions of the tube 20a and the tube 45 are in tight contact with the side surface of the cutter 55, so that outside air does not enter the inside of the tube 20a and the tube 45. As shown in FIG.

As shown in FIG. 9(c), while keeping the melted and cut portions of the tube 20a and tube 45 in close contact with the side surface of the cutter 55, at least one of the holders 121 and 132 is moved to Ensure that each fused cut portion of 45 is co-linear.

As shown in FIGS. 10(a) and 10(b), the cutter 55 between the respective melt-cut portions of tube 20a and tube 45 is removed while the melt-cut portions of tube 20a and tube 45 are removed. Join the parts together. As a result, the tube 20a and the tube 45 can be joined aseptically without allowing outside air to enter the inside of the tube 20a and the tube 45. FIG. After that, the tube 20a and the tube 45 are removed from the holders 121,132.

The method of connecting the tube 20a and the tube 45 is not particularly limited, and for example, the tube 20a and the tube 45 may be connected using a sterile connector.

By joining tube 20a and tube 45, the substance contained in tube 45 comes into contact with the cells contained in tube 20a. Examples of substances contained in tube 45 include inducers, cryopreservation media, media, buffers, immunostaining reagents, nucleic acid extraction reagents, and protein extraction reagents.

As shown in FIG. 11, a section 20a of tube 20 closed at both ends may be placed in a container 40 having a cap 41 therein. After putting the portion 20a of the tube 20 into the container 40, the cap 41 is sealed. Examples of materials for the container 40 include glass and resin. The container 40 may be made of a material that can be penetrated by an injection needle. The cap 41 is made of, for example, a resin such as rubber through which an injection needle can pass. The container 40 is, for example, superior to the tube 20 in at least one of pressure resistance, weather resistance, heat resistance, and cold resistance. Therefore, even if the tube 20 is damaged inside the container 40 , it is possible to prevent the cells inside the tube 20 from diffusing out of the container 40 . In addition, contamination of the cells in the tube 20 with substances outside the container 40 can be suppressed. Examples of tubes 20 include cryotubes.

When the cells are dispersed in the cryopreservation medium in the portion of the tube 20 with both ends closed, the cells and the cryopreservation medium in the portion of the tube 20 may be frozen. The cells and the cryopreservation solution in the tube 20 portion may be frozen while the tube 20 portion is placed in the container 40 . After freezing, the cells may be thawed and recovered from tube 20 . When collecting the cells from the tube 20, the tube 20 may be pierced with an injection needle and the cells may be aspirated with a variable volume aspirator such as a syringe. In addition, it is not always necessary to freeze the cells sucked from the cell incubator 10 into the tube 20 . For sampling purposes, cells may be aspirated from cell incubator 10 into tube 20 and the aspirated cells analyzed. For example, cells aspirated from cell incubator 10 into tube 20 may be analyzed molecularly and/or cytobiologically. For example, cells may be analyzed by immunostaining or by flow cytometry. Nucleic acids contained in cells may be analyzed by PCR or a sequencer. The genome contained in the cell may be sequenced, or the RNA may be sequenced. The epigenetic state of cells may be analyzed. Cell proteins may be analyzed. The tube 20 may contain in advance a reagent that reacts with the cells. As a result, when cells are sucked from the cell incubator 10 into the tube 20, the reagent in the tube 20 reacts with the cells. Examples of reagents include immunostaining reagents, nucleic acid extraction reagents, and protein extraction reagents.

(Second embodiment)
The cell incubator 10 of the cell collection kit according to the second embodiment shown in FIG. 12 includes a housing 100 in which a culture chamber 110 is provided, as shown in FIG. The housing 100 is provided with a first hole 111 and a second hole 112 that connect the outside of the housing 100 and the culture chamber 110 . Cells are cultured in the culture chamber 110 of the housing 100 . Incubation chamber 110 is hollow. The size and shape of the culture chamber 110 are not particularly limited as long as cells can be cultured inside.

The housing 100 is configured, for example, so that the inside can be closed from the outside air. The material of the housing 100 is selected, for example, so that the interior does not exchange gases, viruses, microorganisms, impurities, etc. with the outside through the walls of the housing 100 . Examples of materials for the housing 100 include resin and glass. The housing 100 may be transparent. For example, the tube 20 is connected to the first hole 111 of the housing 100 . For example, the tube 25 is connected to the second hole 112 of the housing 100 . Tubes 20 , 25 may be sterilely joined to housing 100 .

A suction device 30 is connected to the first hole 111 of the housing 100 via the tube 20 . A syringe 35 , empty or containing any fluid, is connected to the second hole 112 of the housing 100 via the tube 25 . Injector 35 has a variable volume. Injector 35 may comprise a syringe and a plunger inserted into and movable within the syringe. Alternatively, syringe 35 may be a flexible bellows or bag.

As shown in FIG. 14, the aspirator 30 is used to aspirate the fluid containing the cells in the cell incubator 10 into the tube 20, and the injector 35 is used to aspirate the fluid in the injector 35 into the cell incubator 10. inject inside. Thereafter, as in the first embodiment, after the fluid containing cells is sucked into the tube 20 , one or more portions of the tube 20 are closed using the tube sealer 80 . Other components of the second embodiment are the same as those of the first embodiment.

(Third embodiment)
As shown in FIGS. 15, 16, and 17, in the method of collecting cells according to the third embodiment, a suction needle is inserted into the bag 20a from outside the container 40 containing the bag 20a containing cells. and aspirating cells into aspirator 51 through aspiration needle 50 . An arbitrary portion of the container 40 may be pierced with the suction needle 50 , and the cap 41 may be pierced with the suction needle 50 .

The bag 20a may be the portion 20a of the tube 20 closed at both ends as described in the first embodiment. The cells in the bag 20a may be cryopreserved before the cells are collected from the bag 20a through the suction needle 50. In this case, after the frozen cells are thawed, the cells are recovered from the bag 20a through the suction needle 50. FIG.

As described in the first embodiment, the cap 41 is made of resin such as rubber through which an injection needle can pass, for example. Therefore, the suction needle 50 can pass through the side surfaces of the cap 41 and the bag 20a.

In inserting the suction needle 50 into the bag 20a from outside the container 40, as shown in FIGS. 18 and 19, a guide 60 for positioning the suction needle 50 on the cap 41 of the container 40 may be used. The guide 60 is provided with a container housing recess 61 for housing the container 40 and the cap 41 and an insertion hole 62 into which the suction needle 50 is inserted.

The container 40 with the cap 41 fixed is inserted into the container storage recess 61 . The container housing recess 61 and the container 40 to which the cap 41 is fixed are fitted, for example. For example, the internal shape of the container storage recess 61 and the external shape of the container 40 to which the cap 41 is fixed are similar.

The insertion hole 62 is connected, for example, to the center of the bottom surface of the container storage recess 61 . By inserting the suction needle 50 into the insertion hole 62 of the guide 60 housing the container 40 to which the cap 41 is fixed, the suction needle 50 can be easily pierced through the cap 41 .

As shown in FIGS. 20 and 21, an injection needle 70 may be inserted into the bag 20a from outside the container 40. FIG. Any portion of the container 40 may be pierced with the injection needle 70 , and the cap 41 may be pierced with the injection needle 70 . When cells are aspirated into the aspirator 51 through the aspiration needle 50, by supplying fluid from the injector 71 into the bag 20a through the injection needle 70, aspiration by the aspirator 51 is facilitated.

DESCRIPTION OF SYMBOLS 10... Cell incubator, 11... Housing, 12... Lid, 13... Filter, 20... Tube, 20a, 20b, 20c, 20d... Tube portion or bag, 21A , 21B, 21C... Obstructed portion, 25... Tube, 30... Aspirator, 35... Injector, 40... Container, 41... Cap, 45... Tube, 50... . Suction needle 51 . Injector, 80 Tube sealer, 100 Case, 110 Culture chamber, 111 First hole, 112 Second hole, 121 Holder

Claims (15)

connecting the tubing to a cell incubator in which the cells are cultured;
aspirating the cells from the cell incubator into the tube;
occluding one or more portions of the tube;
A method of collecting cells, comprising: The method for collecting cells according to claim 1, wherein the cells are in a cryopreservation solution. 2. The method of recovering cells according to claim 1, wherein the cells are in a medium or a buffer. 4. The method of recovering cells according to any one of claims 1 to 3, further comprising counting the number of said cells in between blocked portions of said tube. 5. The method of collecting cells according to any one of claims 1 to 4, further comprising cutting the blocked portions of the tube to obtain portions of the tube having both ends blocked. 6. The method of recovering cells of claim 5, further comprising counting the number of said cells in said portion of tube. 10. The method of claim 1, further comprising calculating the number of tube segments required based on the number of cells in the tube segment, the length of the tube segment, and the number of cells required. 7. The method for collecting cells according to 6. 8. The method of collecting cells according to any one of claims 5 to 7, further comprising placing the tube portion in a container. 9. The method of recovering cells of any one of claims 5-8, further comprising freezing the liquid within the portion of the tube. sticking an aspiration needle into the bag from the outside of the container containing the bag containing the cells;
aspirating the cells through the aspiration needle;
A method of collecting cells comprising: sticking an injection needle into the bag from outside the container;
introducing fluid into the bag through the needle;
The method of recovering cells according to claim 10, further comprising: The cell recovery method according to claim 10 or 11, wherein a guide for positioning the suction needle in the container is used in inserting the suction needle into the bag from the outside of the container. a cell incubator;
a tube connected to the cell incubator;
an aspirator for aspirating cells in the cell incubator into the tube;
a tube sealer that closes the tube;
A cell recovery kit, comprising: 14. The cell collection kit of claim 13, wherein the tube is marked with a closed position. 15. The cell collection kit according to claim 13 or 14, further comprising a container containing the portion of the tube that has been blocked and cut.

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