JP2009261329A - Cell separation device - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a technique relating to a cell separation device using a mesh, which can be used more easily, as compared with conventional devices and having excellent durability. <P>SOLUTION: The cell separation device is provided with: a mesh-formed separation part for disintegrating a cell aggregate to separate a recovering cell from the cell aggregate and having a strength sufficient for the disintegration of the cell aggregate; a holding part to hold the outer edge of the separation part and suppress the deformation of the separation part in the case of disintegrating the cell aggregate and separating the recovering cell from the cell aggregate; and a gripping part connected to the holding part and gripped by the user. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a cell separation device.

In research and development conducted in the medical field, cells are prepared daily from organs. For the disruption of organs and the isolation of leukocytes from organs, which are performed when cells are prepared from organs, conventionally, a so-called tea strainer is substituted, or a stainless steel mesh cut to an appropriate size is used. Used, or nylon mesh. As a technique for separating specific cells from a cell population, for example, a technique described in Patent Document 1 is known.
JP 2001-178 A

  Conventionally, so-called tea strainers, stainless steel meshes, or nylon meshes have been used to disrupt organs or isolate cells such as leukocytes from organs. The so-called tea strainer is a substitute and is not originally intended for crushing organs, so it is unsuitable for isolating leukocytes with a complete and high survival rate. In addition, tea strainers are not structured to withstand autoclaving and are therefore unsuitable for repeated use over a long period of time due to corrosion. Stainless steel mesh is disposable, and it is necessary to cut the mesh and attach it to the saucer each time, so it is difficult to say that it is a simple method, and it is not suitable for crushing large organs such as the liver. . Nylon meshes are generally widely used, but cannot be reused, lack durability, and cannot crush large organs such as the liver. Therefore, it is desired to develop a cell crushing apparatus that has excellent durability and can crush relatively large organs such as the liver. On the other hand, in recent years, interest in “Earth-friendly products” has increased, and in the medical field, disposable products that become medical waste are gradually being replaced by reusable products, which have excellent environmental characteristics. There is a need for a cell separator.

  In view of the above-described background, an object of the present invention is to provide a technique related to a cell separation apparatus using a mesh, which is easier to use than conventional ones and excellent in durability.

  In the present invention, the following means are adopted in order to solve the above-described problems. That is, the cell separation device according to the present invention is a mesh-like separation unit that crushes a cell aggregate and separates recovered cells from the cell aggregate, and has sufficient strength to crush the cell aggregate. A holding portion that is connected to an outer edge portion of the separation portion and holds the separation portion, which suppresses deformation of the separation portion when crushing the cell aggregate and separating the collected cells from the cell aggregate. And a gripping part connected to the holding part and gripped by the user.

  According to the present invention, since the separation unit has sufficient strength to crush the cell aggregate, and since such a separation unit is held by the holding unit, the cell separation device having excellent durability. Can be provided. In addition, the cell separation device according to the present invention has a gripping part connected to the holding part and is excellent in usability.

The separation unit crushes the cell aggregate and separates the recovered cells. Examples of cell aggregates include various organs such as humans, mice, and rats, which are appropriately selected according to the purpose of research and development. The collected cells are cells separated from cell aggregates such as organs, and examples include leukocytes and hepatocytes.
Recovered cells are also appropriately selected according to the purpose of research and development, and desired recovered cells can be separated by appropriately changing the size of the mesh according to the purpose of research and development. In addition, the separation unit according to the present invention has sufficient strength to crush cell aggregates, and can crush organs that cannot be crushed by so-called tea strainers that have been used as substitutes in the past. is there. The material of the separation part is not particularly limited, but, for example, by using a metal, it is possible to ensure the strength required for crushing the cell aggregate.

  Here, when the cell aggregate is crushed, the cell aggregate is pressed in a direction substantially orthogonal to the surface of the separation part having a mesh. That is, when the cell aggregate is crushed, a predetermined force is applied to the mesh. If the separation part is deformed when the cell aggregate is pressed, the cell aggregate may not be effectively crushed. Therefore, in the cell separation device according to the present invention, the outer edge portion of the separation portion is held by the holding portion. Thereby, the deformation | transformation of the isolation | separation part which may occur when crushing a cell aggregate and isolate | separating a collection | recovery cell from this cell aggregate is suppressed. That is, by connecting the outer edge part of the separation part to the holding part, when the cell aggregate is pressed against the separation part, a tensile force acts on the separation part, and bending is reduced. As a result, the cell aggregate can be more reliably separated. In the prior art, it is necessary to cut the stainless steel mesh each time and attach it to the saucer, which requires troublesome work. Also, if the attachment state to the saucer is insufficient, crushing cannot be performed reliably. There was also concern. On the other hand, according to the cell separation device of the present invention, such a problem can be solved.

  The grip portion is connected to the holding portion and is gripped by a user (a person who uses the cell separation device). The shape of the grip portion is not particularly limited, but it is preferable that the grip portion has a length that can be stably gripped by the user, and the operability can be further improved by using a length that can be stably gripped. Moreover, it can be set as the holding part which is easier to hold | grip by giving a certain width and thickness to a holding part. In addition, since the conventional stainless steel mesh or nylon mesh that is cut each time is not provided with a gripping portion, the separation portion must be gripped directly, resulting in poor operability. Moreover, in such a conventional mode, there is a concern that microorganisms present in the air are mixed. On the other hand, the cell separation device according to the present invention includes a gripping portion having a certain length, thereby ensuring a distance from the separation portion to a portion gripped by the user. As a result, the cell aggregate can be crushed in a state in which the periphery of the separation unit is isolated from the outside so that microorganisms in the air do not enter the collected cells. That is, the aggregated cell body can be handled aseptically. Therefore, you may design the holding | grip part which concerns on this invention in the length which can suppress mixing of the microorganisms which exist in the air.

  Here, in the cell separation apparatus described above, a plurality of wire members constituting the mesh are configured to suppress damage to the collected cells when the cell aggregates are crushed and the collected cells are separated from the cell aggregates. Each preferably has a chamfered structure. Thereby, according to the cell separation device concerning the present invention, it becomes possible to prevent the recovery cell from being damaged when the recovery cell is separated. As a result, for example, when leukocytes are prepared as recovered cells, leukocytes with a very high survival rate can be prepared.

  Further, in the above-described cell separation device according to the present invention, the separation part is bent at an outer edge part, and the holding part includes a first ring member to which the grip part is connected and an inner diameter of the first ring member. A second ring member having a slightly smaller outer diameter, and sandwiching the bent outer edge between the inner surface of the first ring member and the outer surface of the second ring member, thereby May be held. With this configuration, it is possible to stably hold the separation unit.

In addition, it is preferable that each structure of the cell separation apparatus which concerns on this invention mentioned above, ie, the said separation part, the said holding | maintenance part, and the said holding part, is comprised with the metal members which can endure an autoclave process. More preferably, the separation part, the holding part, and the grip part are made of stainless steel. By making it metal, durability can be improved compared with the conventional nylon mesh. In addition, the autoclave treatment can be reused, and a cell separation apparatus excellent in environmental performance can be provided. In addition, the cell separation device according to the present invention is hardly corroded with respect to various disinfectants and can be subjected to flame sterilization. Furthermore, according to the cell separation device of the present invention, since it has sufficient strength and durability, not only relatively small organs such as the spleen but also relatively large organs such as the liver can be crushed in a short time. Can do.

  ADVANTAGE OF THE INVENTION According to this invention, it is a cell separation apparatus using a mesh, Comprising: The technique regarding the cell separation apparatus which was easier to use than before, and was excellent in durability can be provided.

  Next, an embodiment of a cell separation device according to the present invention will be described based on the drawings.

<Configuration>
FIG. 1 shows a plan view of the cell separation device according to the embodiment, and FIG. 2 shows a side view of the cell separation device according to the embodiment. FIG. 3 is an exploded perspective view of the cell separation device according to the embodiment. As shown in these drawings, the cell separation device 100 according to the embodiment includes a mesh 1, a first ring 2, a second ring 3, and a grip portion 4. Each configuration will be described below.

  The mesh 1 corresponds to a separation unit of the present invention, and crushes cell aggregates such as various organs such as humans, mice, and rats, and separates recovered cells (for example, white blood cells). Since cell aggregates and recovered cells are appropriately selected according to the purpose of research and development, the size of the mesh and the type of mesh may be prepared based on the purpose of research and development. The mesh 1 according to the present embodiment includes a bottom 11 that separates cell aggregates, and a connection portion 12 (see FIG. 3) that is sandwiched between the first ring 2 and the second ring 3. The bottom portion 11 of the present embodiment is circular and is curved in the direction in which the cell aggregate is inserted, and is devised so that crushing and separation can be performed more reliably. Moreover, the connection part 12 is formed by bending the outer edge part of the bottom part 11 upwards, and the connection part 12 and the bottom part 11 are formed integrally.

  In this embodiment, the mesh 1 is made of stainless steel, but the material, the diameter of the wire constituting the mesh, and the degree of mesh may be appropriately selected according to the organ to be crushed and the cells to be collected. For example, nickel, monel, brass, red brass, phosphor bronze, copper, iron, galvanized wire, steel, aluminum, titanium, nichrome, hastelloy, inconel, etc. can be used as the material. Examples of stainless steel include SUS304, SUS316, SUS316L, SUS430, and SUS310S.

The wire diameter is preferably 0.02 mm to 2.00 mm, which is usually used as a woven wire mesh.
The mesh is a mesh (number of meshes between 25.4 mm on one side) or an open mesh (space). The opening (mm) is calculated by Equation 1, and the space ratio (%) is calculated by Equation 2.
Opening = 25.4 / mesh-wire diameter: Formula 1
Space ratio = (opening) ² / (opening + wire diameter) ² × 100 Formula 2
The degree of mesh may be selected according to the cell type, but 50 to 80 mesh is preferable.

The mesh may be formed by pressing small holes in a thin metal plate, but is preferably produced by weaving a metal wire such as stainless steel. For weaving, weaving is plain weaving (PLAIN WEAVE), twill weaving (TWILLED WEAVE), twisted weaving (STAND WEAVE), Aya Sugi (HERRINGBONE TWILLED WEAVE), satin weaving (SATIN WEAVE), flat weaving (PLAIN DUTCH WEAVE), twill Tatami weave (TWILLED DUTCH
WEAVE), REVERSE DUTCH WEAVE, STRAND TWILLED WEAVE, TRIPLE WARP WEAVE, and TIRE FABRIC WEAVE.

  Here, FIG. 4 shows an enlarged view of the mesh 1 when the mesh 1 is a plain weave. FIG. 5 shows an enlarged view of the mesh 1 when the mesh 1 is a twill weave. The plain weave is a standard weaving method of the woven wire mesh, and the vertical lines and the horizontal lines are arranged at a predetermined interval and are configured to cross each other one by one. Twill weave is used when weaving a relatively high mesh or a wire mesh with a large wire diameter for a mesh that is difficult to weave with a plain weave. In twill weaving, vertical lines and horizontal lines are arranged at regular intervals and cross over each other by two or more. Moreover, in this embodiment, the vertical line and horizontal line which comprise a mesh are chamfered. This makes it possible to prevent damage to the collected cells when separating the collected cells. As a result, for example, when leukocytes are prepared as recovered cells, leukocytes with a very high survival rate can be prepared.

  The first ring 2 and the second ring 3 correspond to the holding portion of the present invention. The 1st ring 2 is comprised by the cyclic | annular stainless steel member, and the holding part 4 is connected to the outer surface. The second ring 3 is also composed of an annular stainless steel member. The outer diameter of the second ring 3 is designed to be slightly smaller than the inner diameter of the first ring 2, and the connecting portion 12 of the mesh 1 is sandwiched between the inner surface of the first ring 2 and the outer surface of the second ring 3. The Thus, when the cell assembly is pressed by holding the connection portion 12 of the mesh 1 by the first ring 2 and the second ring 3, a tensile force acts on the mesh 1 and the deflection is reduced. As a result, the cell aggregate can be more reliably separated. The first ring 2 and the second ring 3 may be configured to be disassembled, or may be fixed with an existing adhesive or the like. According to the former, the mesh 1 can be attached and detached, and according to the latter, the mesh 1 can be held more stably.

  The grip portion 4 is made of a rod-shaped stainless steel member and is connected to the outer surface of the first ring 2. The grip portion 4 of the present embodiment is bent so that one stainless rod-shaped member is folded at a substantially center, and the interval between the parallel rod-shaped members from the folded free end portion 41 toward the connection end portion 42 is increased. Designed to gradually widen. The free end portions 42 of the parallel bar-like members are welded to the outer surface of the first ring 2. Further, a bent portion 43 is provided in the vicinity of the free end portion 41. The cell separation device 100 of the present embodiment is more easily gripped by bending the free end 41 side of the grip portion 4 so that the free end 41 points obliquely downward with the bent portion 43 as a base point. It has been devised. The grip 4 is designed to have a length that can be stably gripped by the user (in this embodiment, the length from the center of the mesh 1 to the free end 41 of the grip 4 is about 140 mm). Furthermore, since the holding part 4 has a sufficient length, according to the cell separation device 100 of the present embodiment, it is possible to aseptically handle the cell aggregate to be destroyed and separated.

<Example>
Next, examples using the cell separation device 100 of the above-described embodiment will be described. In the examples described below, a plain weave mesh (see FIG. 4) was used for the cell separation device 100. Moreover, SUS304 was employ | adopted for the material of the mesh 1, and the diameter of the bottom part 11 of the mesh 1 was 50 mm.

(Facility / equipment)
Venue: Gunma University School of Health, Central Building, 5th floor, Infection / Immune System Exercise Room, Infection / Immune System Laboratory, Infection / Immunology / Blood Laboratory Room: Clean Bench (Hitachi / CCV-1301EC), Safety Cabinet (Showa) Science / SEC2A-1300V), Centrifuge (Beckman / Allegra-GKR)
Venue: Gunma University School of Medicine, Clinical Research Building, 4th floor Organ Pathology Control Pathology Tumor Control Laboratory Tumor Radiology Equipment: Flow cytometer (FACSCallibur) (BD Biosciences)

(antibody)
FITC-labeled CD3ε (145-2C11), biotin-labeled NK1.1 (PK136), and Fcγ-receptor (R)
A monoclonal antibody (mAb) against (2.4G2) purchased from BD PharMingen (Hamburg, Germany) was used for the experiment.

(reagent)
Fetal bovine serum (FCS) is Trace Biosciences (Castle Hill, HSW, Australia), Percoll (specific gravity: 1.124 g / ml) is Biochrom (Berlin, Germany), RPMI 1640 is NISUI PHAMACEUTICAL CO., LTD (Tokyo, Japan), HEPES is from DOJINDO LABORATORIES (Kumamoto, Japan), L-glutamine and penicillin / streptomycin are from Invitrogen (Carlsbad, CA, USA), 2-mercaptoethanol, sodium hydrogen carbonate, ammonium chloride, TRIS and sodium azide are from Wako Pure Chemical Industries, Ltd. (Osaka, Japan), Bovine serum albmin (BSA) from Thermo (Hamilton, New Zealand), streptavidin (SA) labeled Cy5 from BD PharMingen, paraformaldehyde (PFA) from Merck (Darmstadt, Germany) Was subjected to the experiment.

(Preparation of intrahepatic leukocytes)
C57BL / 6 mice (8-12 weeks old male or female) purchased from Charles River Japan (Yokohama), bred and bred in a specific pathogen-free environment at the Gunma University School of Medicine Animal Experiment Facility or cervical dislocation or diethyl ether The liver was removed after euthanization. Liver is complete medium (CM: 20 mM HEPES, 10% sodium hydrogen carbonate, L-glutamine (293 μg / ml), penicillin (100 U / ml), streptomycin (100 μg / ml), FCS (10%), 2-mercaptoetanol ( After perfusion with a 50 mM) -containing RPMI 1640 medium), the cells were separated by the cell separation apparatus 100 (plain weave mesh, SUS304) of the above-described embodiment, and the cell suspension was centrifuged at 500 rpm for 20 seconds. The supernatant was centrifuged at 1500 rpm for 5 minutes, 40% percoll (diluted with CM) was added to the sediment, and the mixture was overlaid with 70% percoll (diluted with CM). After centrifugation at 1800 rpm for 23 minutes, an intermediate layer of 40% and 70% percoll was recovered. After hemolysis with Lysis buffer (8.3 mg NH ₄ Cl dissolved in distilled water and 111 ml 0.17 M Tris buffer (pH 7.65)), the cells were washed with CM and the cells were adjusted to 1 × 10 ⁶ / ml. Floating on CM.

(Flow cytometry)
In order to prevent non-specific binding of the antibody, the cells were reacted with anti-FcγR (2.4G2) mAb at 4 ° C. for 15 minutes and then reacted with FITC / Biotin-labeled mAb at 4 ° C. for 15 minutes. Biotin-labeled antibodies were visualized with SA-labeled Cy5 (BD ParMingen). After staining, the cells were fixed with 1% PFA-containing PBS (−), taken up with FACSCalibur (BD PharMingen), and analyzed with CellQuest software (BD PharMingen).

(effect)
Here, Table 1 shows a comparison table of the number of recovered cells in the conventional nylon mesh and this example. FIG. 6 shows the results of the flow cytometer according to this example. Table 2 shows a comparison table between the conventional nylon mesh and the cell separation device of this example. As shown in Table 1, the number of recovered cells in this example exceeds the number of recovered cells by the conventional nylon mesh. In addition, as shown in FIG. 6, it can be seen that T cells, NKT cells, and NK cells are separated very efficiently by using the cell separation device 100. In addition, as shown in Table 2, the cell separation device of the example has superior effects in terms of size, reusability, durability, ease of use, and cell recovery rate as compared with the conventional nylon mesh. Play. Regarding economic efficiency (cost),
Since the cell separation device of the present embodiment is reusable, it is possible to reduce the running cost.

  According to the cell separation device 100 of the embodiment described above, convenience, workability, and durability can be improved. More specifically, the durability can be improved as compared with a conventional nylon mesh. In addition, by applying autoclaving, it is possible to provide a cell separation device that can be reused and is excellent in environmental performance. Further, the cell separation device 100 is hardly corroded against various disinfectants and can be subjected to flame sterilization. Furthermore, since the cell separator 100 has sufficient strength and durability, not only a relatively small organ such as the spleen but also a relatively large organ such as the liver can be crushed in a short time.

In addition, the cell separation device of the embodiment is used in many facilities as a simple tool for minimizing the risk of microbial contamination, crushing organs easily and efficiently, and recovering cells with high survival rate. Can do. In the medical field including research institutions, it is difficult to advance research and development without preparing cells from organs. Therefore, the cell separation device can be widely used in medical facilities such as hospitals, clinical laboratory contract centers, medical universities and research institutions, and companies such as pharmaceutical companies.

  Although the preferred embodiments of the present invention have been described above, the cell separation device according to the present invention is not limited to these, and can include combinations thereof as much as possible.

The top view of the cell separation device concerning an embodiment is shown. The side view of the cell separation device concerning an embodiment is shown. The disassembled perspective view of the cell separation device concerning an embodiment is shown. An enlarged view of the mesh when the mesh is a plain weave is shown. An enlarged view of the mesh when the mesh is a twill weave is shown. The result of the flow cytometry by a present Example is shown.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ... Mesh 2 ... 1st ring 3 ... 2nd ring 4 ... Grasp part 11 ... Bottom part 12 ... Connection part 41 ... Free end part 42 ... Connection end part 43 ... Bending part 100 ... Cell separation device

Claims (4)

A mesh-like separation unit for crushing the cell aggregate and separating the recovered cells from the cell aggregate, the separation unit having sufficient strength to crush the cell aggregate;
A holding part for holding the outer edge part of the separation part, which suppresses deformation of the separation part when crushing the cell aggregate and separating the recovered cells from the cell aggregate;
A gripping part connected to the holding part and gripped by a user;
A cell separation apparatus comprising:   Each of the plurality of line members constituting the mesh-like separation unit is configured to suppress damage of the collected cells when the separated cells are crushed and the collected cells are separated from the cell aggregate. The cell separation device according to claim 1, which has a chamfered structure. The separating portion is bent at the outer edge,
The holding portion includes a first ring member to which the grip portion is connected, and a second ring member having an outer diameter slightly smaller than the inner diameter of the first ring member, and an inner surface of the first ring member; The cell separation device according to claim 1 or 2, wherein the separation part is held by sandwiching the bent outer edge part between the outer side surface of the second ring member.   The cell separation device according to any one of claims 1 to 3, wherein the separation unit, the holding unit, and the gripping unit are made of a metal member that can withstand autoclaving.

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