JP2023096594A - Cell trapping device, cell trapping apparatus and cell trapping method - Google Patents

Abstract

To provide a cell trapping device, a cell trapping apparatus and a cell trapping method which can trap a target cell in a test solution more efficiently.SOLUTION: A cell trapping device comprises a first space, a second space, an inlet connected to the first space, an outlet connected to the second space, a filtration part which connects the first space to the second space and blocks the passage of particles greater than a predetermined size, and a cell adsorption layer which is at least in contact with the first space and adsorbs a target cell in a test solution.SELECTED DRAWING: Figure 1

Description

TECHNICAL FIELD The present application relates to a cell-trapping device, a cell-trapping apparatus and a cell-trapping method.

Intravascular fluids such as blood and lymph contain various cells in addition to blood cell components and plasma components. In recent years, it has been studied to detect and separate cells contained in these intravascular fluids and use them for the prevention and treatment of diseases. For example, certain tumor cells break free from the primary tumor tissue, enter the blood, and exist in the blood as Circulating Tumor Cells (sometimes abbreviated as CTCs). By detecting circulating tumor cells in blood and estimating the amount in blood, it is used to predict cancer progression and metastasis.

If the cells in the intravascular fluid can be separated without damage, it will be possible to proliferate the cells and analyze the information held by the cells, which is expected to contribute to the development of medicine and medical technology. For example, Patent Literature 1 discloses a method of adsorbing and separating cells in a test fluid using a hydratable composition containing intermediate water.

Japanese Patent No. 6468520

In general, there are very few circulating tumor cells in the blood. For this reason, it is preferable that the cells in the intravascular fluid can be trapped as efficiently as possible. The present disclosure provides a cell trapping device, a cell trapping apparatus, and a cell trapping method capable of trapping target cells in a test fluid more efficiently.

A cell trapping device according to an embodiment of the present disclosure includes a first space, a second space, an inlet connected to the first space, an outlet connected to the second space, and the first a filtering unit that connects the space and the second space and suppresses the passage of particles larger than a predetermined size; and a cell adsorption unit that is disposed in contact with at least the first space and adsorbs target cells in the test fluid. a layer;

The present disclosure provides a cell trapping device, a cell trapping apparatus, and a cell trapping method capable of efficiently trapping target cells in a test fluid.

1 is an exploded perspective view of a cell trapping device according to a first embodiment; FIG. 2A is a top view of the cell trapping device shown in FIG. 1. FIG. FIG. 2B is a cross-sectional view of the cell trapping device taken along line 2B-2B of FIG. 2A. 3A is a top view of a cell trapping device according to another form of the first embodiment; FIG. FIG. 3B is a cross-sectional view of the cell trapping device taken along line 3B-3B of FIG. 3A. FIG. 4 is a schematic diagram illustrating trapping of target cells using a cell trapping device. FIG. 5 is a schematic diagram illustrating trapping of target cells using a cell trapping device. FIG. 6 is an exploded perspective view of a cell trapping device according to a second embodiment; 7A is a top view of the cell trapping device shown in FIG. 6. FIG. FIG. 7B is a cross-sectional view of the cell trapping device taken along line 7B-7B of FIG. 7A. FIG. 8 is a schematic diagram showing the configuration of the cell trapping device according to the third embodiment. FIG. 9 is a schematic diagram explaining the operation of the cell trapping device. FIG. 10 is a schematic diagram explaining the operation of the cell trapping device. FIG. 11 is a schematic diagram explaining the operation of the cell trapping device.

（式（１）において、Ｒ^１は、水素原子またはメチル基であり、Ｒ^２は、メチル基またはエチル基であり、ｍは１以上３以下の整数であり、ｎは繰り返し単位である。）

（式（２）において、Ｒ１は、水素原子またはメチル基であり、ｎは繰り返し単位である。）

（式（３）において、Ｘ、Ｙは、それぞれ独立に水素原子、アルカリ金属原子、又は、アンモニウム基であり、ｍは０以上以下の整数であり、ｎは繰り返し単位である。）

（式（４）において、Ｒ^１は水素原子または炭素数１以上３０以下の有機基であり、ｎは繰り返し単位である。）
［項目１６］
項目１から１５のいずれか１項に記載の細胞捕捉デバイスと、
前記細胞捕捉デバイスの前記導入口または前記排出口の少なくとも一方に接続されたポンプと、
を備えた、細胞捕捉装置。
［項目１７］
前記ポンプは、双方向ポンプである、項目１６に記載の細胞捕捉装置。
［項目１８］
被検液を保持する被検液保持チャンバーと、
培地を保持する培地保持チャンバーと、
廃液を保持する第１回収チャンバーと、
廃液を保持する第２回収チャンバーと、
洗浄液を保持する洗浄液保持チャンバーと、
前記被検液保持チャンバー、前記培地保持チャンバーおよび前記第１回収チャンバーのうちの１つと、前記導入口とを選択的に接続する第１弁と、
前記第２回収チャンバーおよび前記洗浄液保持チャンバーのうちの１つと、前記排出口とを選択的に接続する第２弁と、
をさらに備える項目１６または１７に記載の細胞捕捉装置。
［項目１９］
被検液から対象細胞を分離する細胞捕捉方法であって、
項目１に記載の細胞捕捉デバイスを用意し、
被検液を前記導入口から導入し、前記排出口から排出させることによって、前記対象細胞を前記細胞吸着層に吸着させる、
細胞捕捉方法。
［項目２０］
前記被検液は他の細胞を含み、
前記吸着させる工程において、前記他の細胞を前記第１空間に保持し、
前記吸着工程後、他の液体を前記排出口から導入し、前記導入口から排出させることによって、前記第１空間内に保持された前記他の液体を前記導入口から排出させる、項目１９に記載の細胞捕捉方法。
［項目２１］
被検液から対象細胞を分離する細胞捕捉方法であって、
項目１８に記載の細胞捕捉デバイスを用意し、
前記被検液保持チャンバーに保持された前記被検液を前記導入口から導入し、前記排出口から排出させて、前記第２回収チャンバーに保持させ、
前記洗浄液保持チャンバーに保持された洗浄液を前記排出口から導入し、前記導入口から排出させて、前記第１回収チャンバーに保持させ、
前記培地保持チャンバーに保持された前記培地を、前記導入口から導入し、前記排出口から排出させて、前記第２回収チャンバーに保持させる、
細胞捕捉方法。 Biological cells are believed to be surrounded by hydration shells composed of water molecules in various states. A hydrophilic material with a similar hydration structure would be preferred. On the other hand, the interaction between such hydrophilic materials and living cells may be weak, making it difficult to trap cells efficiently. The inventor of the present application has come up with a cell trapping device, a cell trapping apparatus, and a cell trapping method that can efficiently trap cells by utilizing the shape of the channel for trapping cells. The cell-trapping device, cell-trapping apparatus, and cell-trapping method of this embodiment are as follows.
[Item 1]
a first space;
a second space;
an inlet connected to the first space;
an outlet connected to the second space;
a filtration unit that connects the first space and the second space and suppresses passage of particles larger than a predetermined size;
a cell adsorption layer arranged in contact with at least the first space and adsorbing target cells in the test solution;
A cell trapping device, comprising:
[Item 2]
The cell trapping device according to item 1, wherein at least one of the first space and the second space is closed except for the introduction port, the discharge port and the filtering section.
[Item 3]
3. According to item 1 or 2, further comprising an upper plate and a lower plate spaced apart from the upper plate by a predetermined gap, wherein the filtering section is formed by the gap between the upper plate and the lower plate. cell trapping device.
[Item 4]
4. A cell trapping device according to item 3, wherein the cell adsorption layer is arranged on the lower plate.
[Item 5]
4. The cell-trapping device of item 3, wherein the gap is a capillary space.
[Item 6]
6. A cell trapping device according to item 5, wherein the gap is 30 μm or less.
[Item 7]
further comprising a plate-shaped spacer having an opening disposed between the upper plate and the lower plate;
The filtering part is formed in the opening of the spacer,
the top plate has an upper surface and a lower surface;
the inlet and the outlet are formed in the upper plate in contact with the upper surface;
The first space and the second space are formed in the upper plate in contact with the lower surface,
4. The cell trapping device of item 3, wherein at least part of the first space and at least part of the second space are in contact with the opening of the spacer.
[Item 8]
8. The cell trapping device according to any one of items 1 to 7, wherein the cell adsorption layer is arranged only in the first space.
[Item 9]
8. The cell trapping device according to any one of items 3 to 7, wherein the cell adsorption layer is arranged on the entire upper surface of the lower plate.
[Item 10]
3. The cell trapping device according to item 1 or 2, further comprising a plate-like filter having a plurality of through-holes, wherein the filtering section is the plurality of through-holes.
[Item 11]
10. The cell trapping device according to item 9, wherein the plurality of through holes have a diameter of 30 μm or less.
[Item 12]
11. The cell trapping device according to item 10, wherein the cell adsorption layer is positioned around the plurality of pores of the filter and is in contact with the first space.
[Item 13]
further comprising a top plate having a top surface and a bottom surface and a bottom plate having a top surface and a bottom surface;
The introduction port is formed in the upper plate in contact with the upper surface,
The first space is formed in the upper plate in contact with the lower surface,
the outlet is formed in the lower plate in contact with the lower surface;
the second space is formed in the lower plate in contact with the upper surface;
13. The cell trapping device of item 12, wherein the filter is positioned between the lower surface of the upper plate and the upper surface of the lower plate.
[Item 14]
The target cells include circulating tumor cells, stem cells, vascular endothelial cells, nerve cells, dendritic cells, smooth muscle cells, fibroblasts, cardiomyocytes, skeletal muscle cells, hepatic parenchymal cells, non-hepatic non-parenchymal cells, and pancreatic cells. 14. The cell trapping device according to any one of items 1 to 13, wherein the cell adsorption layer comprises at least one selected from the group consisting of islet cells, and the cell adsorption layer adsorbs the target cells.
[Item 15]
the cell adsorption layer comprises a first biocompatible polymeric material,
The first biocompatible polymeric material is any one or more of the polymers represented by the following formulas (1) to (4), or a monomer constituting the polymer represented by the formula (1) , polyethylene glycol, polyvinyl pyrrolidone, polyvinyl methyl ether, polymethoxyethyl acrylamide, polymethoxyethyl methacrylamide, polymethoxyethyl vinyl ether, polytetrahydrofurfuryl acrylate and polytetrahydrofurfuryl methacrylate. 15. The cell trapping device according to any one of items 1 to 14, comprising a copolymer with a monomer constituting

(In formula (1), R ¹ is a hydrogen atom or a methyl group, R ² is a methyl group or an ethyl group, m is an integer of 1 or more and 3 or less, and n is a repeating unit.)

(In Formula (2), R1 is a hydrogen atom or a methyl group, and n is a repeating unit.)

(In formula (3), X and Y are each independently a hydrogen atom, an alkali metal atom, or an ammonium group, m is an integer of 0 or more and n is a repeating unit.)

(In formula (4), R ¹ is a hydrogen atom or an organic group having 1 to 30 carbon atoms, and n is a repeating unit.)
[Item 16]
a cell trapping device according to any one of items 1 to 15;
a pump connected to at least one of the inlet or the outlet of the cell trapping device;
A cell trapping device.
[Item 17]
17. A cell trapping device according to item 16, wherein the pump is a bi-directional pump.
[Item 18]
a test liquid holding chamber that holds the test liquid;
a medium holding chamber for holding medium;
a first collection chamber holding a waste liquid;
a second collection chamber for holding waste fluid;
a cleaning solution holding chamber for holding a cleaning solution;
a first valve selectively connecting one of the test solution holding chamber, the medium holding chamber and the first collection chamber to the introduction port;
a second valve selectively connecting one of the second collection chamber and the wash solution holding chamber to the outlet;
18. A cell trapping device according to item 16 or 17, further comprising:
[Item 19]
A cell capturing method for separating target cells from a test solution,
preparing the cell-trapping device according to item 1,
Adsorbing the target cells to the cell adsorption layer by introducing a test solution from the inlet and discharging it from the outlet;
Cell capture method.
[Item 20]
The test solution contains other cells,
holding the other cell in the first space in the adsorbing step;
Item 20. According to item 19, after the adsorption step, another liquid is introduced from the discharge port and discharged from the introduction port, thereby discharging the other liquid held in the first space from the introduction port. cell capture method.
[Item 21]
A cell capturing method for separating target cells from a test solution,
preparing the cell-trapping device according to item 18,
introducing the test liquid held in the test liquid holding chamber from the introduction port, discharging it from the discharge port, and holding it in the second recovery chamber;
introducing the cleaning liquid held in the cleaning liquid holding chamber from the discharge port, discharging it from the introduction port, and holding it in the first collection chamber;
The medium held in the medium holding chamber is introduced from the inlet, discharged from the outlet, and held in the second collection chamber.
Cell capture method.

The cell-trapping device, cell-trapping apparatus, and cell-trapping method of this embodiment trap target cells in a test fluid such as blood or lymph. The target cells are circulating tumor cells, stem cells, vascular endothelial cells, nerve cells, dendritic cells, smooth muscle cells, fibroblasts, cardiomyocytes, skeletal muscle cells, and liver parenchyma cells contained in intravascular fluids such as blood and lymph. It is at least one selected from the group consisting of cells, non-parenchymal liver cells, and pancreatic islet cells. Hereinafter, the cell-trapping device, cell-trapping apparatus, and cell-trapping method of this embodiment will be specifically described with reference to the drawings.

(First embodiment)
FIG. 1 is an exploded perspective view of a cell trapping device 101 of this embodiment, and FIG. 2A is a top view of the cell trapping device 101. FIG. FIG. 2B is a cross-sectional view of cell trapping device 101 taken along line 2B-2B of FIG. 2A.

The cell trapping device 101 includes a first space 11, a second space 12, an inlet 13 connected to the first space 11, an outlet 14 connected to the second space 12, a first space 11 and a second space. A filtering unit 22 that connects the second space 12 and suppresses the passage of particles larger than a predetermined size, and a cell adsorption layer 30 that is arranged in contact with at least the first space 11 and adsorbs target cells in the test fluid. and In this embodiment, the cell trapping device 101 further includes an upper plate 10, a spacer 20, and a lower plate 40 to form the first space 11, the second space 12, and the filtering section 22. .

The upper plate 10 has an upper surface 10a and a lower surface 10b. A recessed portion 10r1 and a recessed portion 10r2 having an opening in the lower surface 10b are provided in the upper plate 10, and the spaces within the recessed portion 10r1 and the recessed portion 10r2 are the first space 11 and the second space 12, respectively. The top plate 10 defines a first space 11 and a second space 12 . The inlet 13 and the outlet 14 are provided on the upper surface 10a of the upper plate 10 and connected to the first space 11 and the second space 12, respectively.

The first space 11 expands the flow path of the test liquid introduced from the inlet 13 and uniformly supplies the test liquid to the filtering section 22 . Also, it provides a space in contact with the cell adsorption layer 30 . As will be described later, in the present embodiment, the filtering section 22 is a slit having an opening extending in the y direction, so the first space 11 has a length approximately equal to that of the filtering section 22 in the y direction. preferably. In addition, it is preferable to have a certain length or more in the x direction in order to increase the space in contact with the cell adsorption layer 30 . It is preferable that the second space 12 also have approximately the same length as the filtering section 22 in the y direction.

In this embodiment, the first space 11 and the second space 12 have a rectangular parallelepiped shape with lengths of, for example, x: 50 mm to 300 mm, y: 50 mm to 300 mm, and z: 10 mm to 50 mm. Also, the openings of the inlet 13 and the outlet 14 have a circular shape with a diameter of, for example, 1 mm to 10 mm.

The lower plate 40 has an upper surface 40a having the same shape and size as the lower surface 10b of the upper plate 10, for example. Lower plate 40 functions as a support that supports other structures of cell trapping device 101 .

The upper plate 10 and the lower plate 40 are made of materials such as resin, metal, glass, and ceramics.

The spacer 20 has, for example, a plate-like shape having approximately the same size as the lower surface 10b of the upper plate 10 . The spacer 20 is a member for arranging the upper plate 10 and the lower plate 40 at a predetermined interval to form the filtering section 22 . The spacer 20 is made of a material such as resin, metal, glass, ceramics, or the like.

The thickness of the spacer 20 is the size of the gap in the filtering section 22 . It is preferable that the gap of the filtering part 22 is a capillary space. Also, the thickness of the spacer 20 should be smaller than the target cells to be captured. For example, the spacer 20 has a thickness of 30 μm or less. Preferably, the spacer 20 has a thickness of 5 μm or more and 10 μm or less. When the thickness is 5 μm or more, cells smaller than the target cells in the sample liquid can pass through the filtering section 22, and the vicinity of the inlet of the filtering section 22 is blocked by cells other than the target cells. can be suppressed. Moreover, since the thickness is 10 μm or less, the target cells can be more reliably retained within the first space 11 .

The spacer 20 is provided with an opening 21 contacting the first space 11 and the second space 12 of the upper plate 10 while the spacer 20 is arranged so as to contact the lower surface 10 b of the upper plate 10 . Opening 21 includes regions 21c and 21e in contact with first space 11 and second space 12, respectively, and region 21d sandwiched between regions 21c and 21e. The region 21d serves as a space for the filtering section 22, as will be described later.

The cell adsorption layer 30 is arranged in contact with the first space 11 . In this embodiment, the cell adsorption layer 30 is supported by the lower plate 40 so as to cover the entire upper surface 40a of the lower plate 40 . The cell adsorption layer 30 may be located only within the first space 11 as shown in FIGS. 3A and 3B.

The cell adsorption layer 30 is a layer that selectively adsorbs target cells in the sample liquid. Cell adsorption layer 30 includes a first biocompatible polymeric material.

The first biocompatible polymeric material may contain intermediate water. It is preferable that the first biocompatible polymeric material can contain intermediate water at a rate of 1 wt % or more and 30 wt % or less with respect to the whole in a hydrated state. In addition, the first biocompatible polymer material preferably contains more intermediate water than free water in the hydrated state.
Specifically, the first biocompatible polymer material includes one or more of polymers represented by the following formulas (1) to (4).

式（１）において、Ｒ^１は、水素原子またはメチル基であり、Ｒ^２は、メチル基またはエチル基であり、ｍは１以上３以下の整数であり、ｎは繰り返し単位である。

In formula (1), R ¹ is a hydrogen atom or a methyl group, R ² is a methyl group or an ethyl group, m is an integer of 1 or more and 3 or less, and n is a repeating unit.

式（２）において、Ｒ^１は、水素原子またはメチル基であり、ｎは繰り返し単位である。

In formula (2), R ¹ is a hydrogen atom or a methyl group, and n is a repeating unit.

式（３）において、Ｘ、Ｙは、それぞれ独立に水素原子、アルカリ金属原子、又は、アンモニウム基であり、ｍは０以上以下の整数であり、ｎは繰り返し単位である。

In formula (3), X and Y are each independently a hydrogen atom, an alkali metal atom, or an ammonium group, m is an integer of 0 or more and n is a repeating unit.

式（４）において、Ｒ^１は水素原子または炭素数１以上３０以下の有機基であり、ｎは繰り返し単位である。

In formula (4), R ¹ is a hydrogen atom or an organic group having 1 to 30 carbon atoms, and n is a repeating unit.

A specific example of the polymer represented by formula (1) is
Poly(2-methoxyethyl acrylate), (PMEA),
Poly(2-ethoxyethyl acrylate), (PEEA or PEtEA),
poly[2-(2-methoxyethoxy)ethyl methacrylate], (PMe2A),
Poly[2-(2-ethoxyethoxy)ethyl acrylate], (PEt2A),
Poly(6-methoxyhexyl acrylate), (PMC6A),
Poly(butoxyethyl acrylate), (PBuEA),
Poly[2-(2-ethoxyethoxy)ethyl acrylate], (PEt2A),
Poly[2-(2-(2-methoxyethoxy)ethoxy)ethyl acrylate] and poly[2-(2-ethoxyethoxy)ethyl methacrylate].

A specific example of the polymeric material represented by formula (2) is poly(tetrahydrofurfuryl acrylate).

Specific examples of polymeric materials represented by formula (3) are polymethylene glutaric acid and polyitaconic acid.

A specific example of the polymeric material represented by formula (4) is poly-α-allyloxymethyl methoxyethyl acrylate.

The first biocompatible polymeric material comprises a monomer constituting the polymer represented by formula (1), polyethylene glycol, polyvinylpyrrolidone, polyvinyl methyl ether, polymethoxyethyl acrylamide, polymethoxyethyl methacrylamide, and polymethoxyethyl vinyl ether. , polytetrahydrofurfuryl acrylate and polytetrahydrofurfuryl methacrylate and a monomer constituting at least one polymer selected from the group consisting of polytetrahydrofurfuryl methacrylate. For example, according to Japanese Patent No. 5975532, the size of target cells that can be adsorbed varies depending on the content of intermediate water that the first biocompatible polymeric material may contain. By varying the proportions of the two or more monomers in the copolymer, it is possible to control the amount of intermediate water that may be included.

The cell trapping device 101 may further include a cell adhesion suppression layer arranged on the inner surfaces of the recesses 10r1 and 10r2. The cell adhesion-suppressing layer suppresses deactivation of target cells in the test liquid by contacting or adsorbing to the inner surfaces of the recesses 10r1 and 10r2 in the first space 11 and the second space 12, respectively. To this end, the cell adhesion-inhibiting layer includes a second biocompatible polymeric material. The second biocompatible polymeric material can contain intermediate water at a rate of 30 wt % or more with respect to the whole in a hydrated state. The second biocompatible polymeric material may contain more free water than intermediate water in its hydrated state. Alternatively, the second biocompatible polymeric material may contain more intermediate water than free water in its hydrated state. The second biocompatible polymeric material is a polymer having phospholipid polar groups. Specifically, the second biocompatible polymeric material includes poly(2-methacryloyloxyethylphosphorylcholine) and the like.

The first biocompatible polymeric material and the second biocompatible polymeric material need only contain intermediate water and/or free water when trapping target cells, and the cell trapping device 101 is produced and obtained. Alternatively, the first biocompatible polymeric material and the second biocompatible polymeric material may not contain intermediate water, free water, or the like during storage or the like.

The cell adsorption layer 30 containing the first biocompatible polymer material and the cell adhesion suppression layer containing the second biocompatible polymer material can be obtained by a coating method such as coating, spray dipping, ultraviolet rays, electron beams, radiation, etc. It can be formed by polymerization, or chemical reaction with a functional group of the material forming the lower plate 40 . Moreover, the thickness of the cell adsorption layer 30 and the cell adhesion suppression layer is, for example, 0.01 μm to 0.4 μm.

As shown in FIGS. 1 and 3, the cell adsorption layer 30 is placed on the upper surface 40a of the lower plate 40, and the spacer 20 is sandwiched between the lower surface of the upper plate 10 and the cell adsorption layer 30. A capture device 101 can be made. The upper plate 10, the spacers 20, the cell adsorption layer 30, and the lower plate 40 may be joined using an adhesive or the like, or the upper plate 10, the spacers 20, the cell adsorption layer 30, and the lower plate 40 may be laminated. A pressure may be applied in the stacking direction from the outside using a clamp or the like. Alternatively, a through-hole may be formed in the laminate composed of the upper plate 10, the spacer 20, the cell adsorption layer 30 and the lower plate 40, and the laminate may be fastened with bolts and nuts.

Next, trapping of target cells using the cell trapping device 101 will be described with reference to FIGS. 4 and 5. FIG. 4 and 5 are schematic diagrams for explaining the operation of the cell trapping device 101. FIG. In cell trapping device 101 , at least one of first space 11 and second space 12 is closed except inlet 13 , outlet 14 and filter section 22 . For example, when the first space 11 is closed except for the region connected to the inlet 13 and the filtering section 22, as shown in FIG. The test liquid is sucked from 201 and introduced into the first space 11 while pressure is applied from the introduction port 13 . The holding chamber 201, the pump 202 and the cell trapping device 101 are connected by suitable flow paths such as resin tubes.

A test liquid is introduced into the first space 11 , passes through the filtering section 22 connected to the first space 11 , and moves to the second space 12 . At this time, the target cells 151 contained in the sample liquid are larger than the gaps of the filtering section 22 , so they cannot pass through the filtering section 22 and remain in the first space 11 . The filtering part 22 is a slit-shaped gap, and the opening connected to the first space 11 is narrow. The flow velocity of the liquid increases, and a large inertial force acts on the target cells 151 contained in the sample liquid, and the target cells 151 are pressed against the cell adsorption layer 30 . As a result, the target cells 151 are efficiently adsorbed to the cell adsorption layer 30 .

The sample liquid contains blood cell components 152 larger than the target cell 151, proteins smaller than the target cell 151, platelets, non-nucleated red blood cells, and the like (hereinafter representatively referred to as protein 153). . Since the blood cell component 152 is also larger than the gap of the filtering section 22 , it cannot pass through the filtering section 22 and remains in the first space 11 . However, even if the blood cell components 152 are pressed against the cell adsorption layer 30 in the first space 11, the interaction between the cell adsorption layer 30 and the blood cell components 152 is weak. not.

Since the protein 153 is smaller than the gap of the filtering section 22 , it passes through the filtering section 22 together with the sample liquid and moves to the second space 12 . In this way, the target cells 151 can be selectively adsorbed to the cell adsorption layer 30 with high efficiency.

On the other hand, if the second space 12 is closed except for the area connected to the outlet 14 and the filtration unit 22, the pump 202 is connected to the outlet 14 of the second space 12, as shown in FIG. . A holding chamber 201 is connected to the inlet 13 of the first space 11 . In order to transfer the sample liquid smoothly, the filtering part 22, the first space 11, the second space 12, the holding chamber 201 and the introduction port 13 are provided so that the air between the holding chamber 201 and the pump 202 can be eliminated. and the channel connecting the pump 202 and the outlet 14 are filled with a liquid such as a culture medium.

When the pump 202 is used to suck the liquid in the second space 12 , the liquid to be tested is introduced into the first space 11 through the inlet 13 by suction force. The test liquid passes through the filtering section 22 connected to the first space 11 and moves to the second space 12 . At this time, the target cells 151 contained in the sample liquid are larger than the gaps of the filtering section 22 , so they cannot pass through the filtering section 22 and remain in the first space 11 . Moreover, the target cell 151 is pressed against the cell adsorption layer 30 in the first space 11 by the change in the flow path described above. As a result, the target cells 151 are efficiently adsorbed to the cell adsorption layer 30 .

The blood cell component 152 cannot pass through the filtering section 22 and stays within the first space 11 . However, even if the blood cell component 152 receives pressure in the first space 11 and is pressed against the cell adsorption layer 30, the interaction between the cell adsorption layer 30 and the blood cell component 152 is weak. 30 is hardly adsorbed. Also, the protein 153 passes through the filtering section 22 together with the sample liquid and moves to the second space 12 . The configuration shown in FIG. 4 also enables the target cells 151 to be selectively adsorbed to the cell adsorption layer 30 with high efficiency.

As described above, according to the cell trapping device 101 of the present embodiment, the test liquid is introduced from the inlet 13 of the cell trapping device 101 and discharged from the outlet 14, thereby allowing the test liquid to pass through the filtering section 22. At this time, the target cells 151 are separated from the sample liquid, and the separated target cells can be efficiently adsorbed to the cell adsorption layer 30 . At least one of the first space 11 and the second space 12 is sealed except for the inlet port 13, the outlet port 14, and the filtering section 22, so that the test liquid is pressure-fed and the flow in the vicinity of the filtering section 22 is controlled. It is possible to press the target cells 151 in the sample liquid against the cell adsorption layer 30 by changing the shape of the channel. Therefore, it is possible to increase the interaction between the target cells 151 and the cell adsorption layer 30 and more efficiently adsorb the target cells 151 to the cell adsorption layer 30 . Therefore, it is possible to capture the target cells 151 in the test fluid more reliably.

(Second embodiment)
FIG. 6 is an exploded perspective view of the cell trapping device 102 of this embodiment, and FIG. 7A is a top view of the cell trapping device 102. FIG. 7B is a cross-sectional view of cell trapping device 102 taken along line 7B-7B of FIG. 7A.

As in the first embodiment, the cell trapping device 102 includes a first space 11, a second space 12, an inlet 13 connected to the first space 11, and an outlet 14 connected to the second space 12. and a filtration unit 22 that connects the first space 11 and the second space 12 and suppresses the passage of particles larger than a predetermined size, and is arranged in contact with at least the first space 11, and the object in the test liquid and a cell adsorption layer 30 that adsorbs cells. In this embodiment, the cell trapping device 101 further includes an upper plate 60, a filter 70, and a lower plate 90 to form the first space 11, the second space 12, and the filtering section 22. .

The upper plate 60 has an upper surface 60a and a lower surface 60b. A recess 60r having an opening in the lower surface 60b is provided in the upper plate 60, and the space inside the recess 60r is the first space 11. As shown in FIG. That is, the first space 11 is formed within the upper plate 60 in contact with the lower surface 60b.

The lower plate 90 has an upper surface 90a and a lower surface 90b. A recess 90 r having an opening in the upper surface 90 a is provided in the lower plate 90 , and the space inside the recess 90 r is the second space 12 . That is, the second space 12 is formed within the lower plate 90 in contact with the upper surface 90a.

An introduction port 13 is provided in the upper plate 60 in contact with the upper surface 60 a , and the introduction port 13 is further connected to the first space 11 . A discharge port 14 is provided in the lower plate 90 so as to be in contact with the lower surface 90b. The outlet 14 is connected with the second space 12 . The opening of the first space 11 on the lower surface 60b of the upper plate 60 and the opening of the second space 12 on the upper surface 90a of the lower plate 90 preferably have the same shape and size.

In this embodiment, the first space 11 and the second space 12 have a rectangular parallelepiped shape with lengths of, for example, x: 50 mm to 300 mm, y: 50 mm to 300 mm, and z: 10 mm to 50 mm.

The filter 70 constitutes the filtering section 22 . Specifically, the filter 70 has a plate shape having approximately the same size as the lower surface 60b of the upper plate 60 and the upper surface 90a of the lower plate 90, for example. The filter 70 has an upper surface 70a and a lower surface 70b, and has a plurality of through holes 70h having openings in the upper surface 70a and the lower surface 70b. A cell adsorption layer 80 is arranged on the upper surface 70a of the filter 70, and through holes 80h are provided at positions corresponding to the plurality of through holes 70h. 70 h of through holes and 80 h of through holes function as filtering part 22 .

The diameter of the through hole 70h is 30 μm or less. Preferably, the diameter of the through hole 70h is 5 μm or more and 10 μm or less. A through hole 70 h of the filter 70 functions as the filtering portion 22 .

The cell adsorption layer 80 contains the first biocompatible polymeric material, as in the first embodiment. The cell adsorption layer 80 is arranged on the upper surface 70a of the filter 70, and has through holes 80h corresponding to the through holes 70h, so that the cell adsorption layer 80 is arranged around the plurality of through holes 70h of the filter 70. The diameter of the through-hole 80h may be the same as the diameter of the through-hole 70h, or may be larger than the diameter of the through-hole 70h.

As shown in FIGS. 6 and 7B, the cell trapping device 102 is fabricated by laminating the cell adsorption layer 30 and the filter 70 between the upper plate 60 and the lower plate 90 in the same manner as in the first embodiment. can do.

In the cell trapping device 102 as well, the filter 70 sandwiched between the first space 11 and the second space 12 functions as the filtering part 22, and the test liquid is rapidly filtered when passing through the filter 70 from the first space 11. Due to the narrowing of the channel, the target cells are pressed against the cell adsorption layer 80 located around the through-holes 70h of the filter 70 . Therefore, the target cells are efficiently adsorbed to the cell adsorption layer 80 as described in the first embodiment.

In addition, by arranging the cell adsorption layer 80 over the entire opening of the first space 11, the cell trapping device 102 can be provided with a larger area of the cell adsorption layer 80, allowing more target cells to be cell-adsorbed. Adsorption with layer 80 is possible.

(Third Embodiment)
Embodiments of cell trapping devices are described. The cell trapping device of the present embodiment can separate target cells from a sample liquid, remove unnecessary cells and the like, purify target cells, and separate target cells in a state more suitable for storage. be. FIG. 8 is a schematic diagram showing one embodiment of the cell trapping device 103. As shown in FIG. The cell trapping device 103 includes a cell trapping device 101, a first pump 211, a second pump 212, a first valve 221, a second valve 222, a test liquid holding chamber 213, a medium holding chamber 214, and a It has a first collection chamber 231 , a second collection chamber 232 and a cleaning liquid holding chamber 215 .

In this embodiment, the cell-trapping device 103 has the cell-trapping device 101 of the first embodiment, but the cell-trapping device 103 may have the cell-trapping device 102 of the second embodiment.

The first pump 211 and the second pump 212 are, for example, bidirectional pumps used for medical treatment or blood analysis. One of the two ports of the first pump 211 is connected to the inlet 13 of the first space 11 and the other is connected to the first valve 221 . Also, one of the two ports of the second pump 212 is connected to the discharge port 14 of the second space 12 and the other is connected to the second valve 222 . In the following description, driving the first pump 211 and the second pump 212 in the “forward direction” is said to move liquid from the first valve 221 through the cell-trapping device 101 in the direction of the second valve 222; Driving the first pump 211 and the second pump 212 in the "reverse direction" is said to move liquid from the second valve 222 through the cell trapping device 101 in the direction of the first valve 221 .

The first valve 221 and the second valve 222 are switching valves. The first valve 221 has four ports P1 to P4, and selectively switches one of ports P1, P2 and P3 to connect to port P4. A first pump 211 is connected to the port P4. The second valve 222 has three ports P1 to P3, and selectively switches between ports P1 and P2 to connect to port P3. A second pump 212 is connected to the port P3. Thereby, the first valve selectively connects one of the test liquid holding chamber 213 , the medium holding chamber 214 and the first recovery chamber 231 to the inlet 13 via the first pump 211 . A second valve also selectively connects one of the second collection chamber 232 and the cleaning solution holding chamber 215 to the outlet 14 via the second pump 212 .

The test liquid holding chamber 213 holds the test liquid and is connected to the port P1 of the first valve 221 . The medium holding chamber 214 holds medium such as tissue culture medium and is connected to the port P2 of the first valve 221 . Also, the first recovery chamber 231 is a chamber for holding waste liquid and is connected to the port P3 of the first valve 221 .

The second collection chamber 232 is a chamber that holds waste liquid and is connected to the port P1 of the second valve 222 . The cleaning liquid holding chamber 215 holds a cleaning liquid such as physiological saline and is connected to the port P2 of the second valve 222 .

The cell trapping device 103 may be configured to operate manually, or may be configured to operate automatically. The cell trapping device 103 further comprises a control circuit 216 for automatic operation of the cell trapping device 103 . Also, the first valve 221 and the second valve 222 are composed of solenoid valves. The control circuit 216 switches the paths of the first valve 221 and the second valve 222 to start and stop the first pump 211 and the second pump 212 in the procedure described below.

Each component described above is connected, for example, by a channel such as a resin tube having an appropriate thickness for transferring the test liquid, culture medium, and washing liquid.

Next, a method for separating target cells using the cell trapping device 103 will be described.

First, with the first valve 221 connecting the test liquid holding chamber 213 and the first pump 211 and the second valve connecting the second pump 212 and the second recovery chamber 232, the first pump 211 and the second recovery chamber 232 are connected. 2 pump 212 is driven in the forward direction. As a result, as shown in FIG. 9, the test liquid flows out from the test liquid holding chamber 213 and is introduced into the first space 11 from the introduction port 13 of the cell trapping device 101 via the first valve 221 and the first pump 211. be done.

As described in the first embodiment, the sample liquid passes through the filtering section 22 and moves to the second space 12 , and the target cells 151 are adsorbed to the cell adsorption layer 30 . Also, the blood cell component 152 larger than the target cell 151 stays within the first space 11 . Protein 153 moves to second space 12 .

Further, the sample liquid containing protein is discharged from the discharge port 14, reaches the second collection chamber 232 via the second pump 212 and the second valve 222, and is held therein.

Next, with the first valve 221 connecting the first collection chamber 231 and the first pump 211 and the second valve connecting the second pump 212 and the washing liquid holding chamber 215, the first pump 211 and the second pump 211 are connected. The pump 212 is driven in the reverse direction. Thereby, as shown in FIG. 10, the washing liquid flows out from the washing liquid holding chamber 215 and is introduced into the second space 12 from the outlet 14 of the cell trapping device 101 via the second valve 222 and the second pump 212 . Furthermore, the cleaning liquid is introduced into the first space 11 through the filtering section 22 .

In the first space 11, the target cells 151 are adsorbed to the cell adsorption layer 30, but the blood cell component 152 is not adsorbed to the cell adsorption layer 30, or only very weak interaction is working. Therefore, the blood cell component 152 is discharged from the first space 11 together with the washing liquid. Furthermore, the washing liquid containing blood cell components reaches the first collection chamber 231 via the first pump 211 and the first valve 221 and is held therein. As a result, contaminants other than the target cells 151 are removed from the first space 11, and the target cells 151 are purified.

Next, with the first valve 221 connecting the medium holding chamber 214 and the first pump 211 and the second valve connecting the second pump 212 and the second collection chamber 232, the first pump 211 and the second Drive the pump 212 in the forward direction. As a result, as shown in FIG. 11, the medium flows out of the medium holding chamber 214 and is introduced into the first space 11 from the introduction port 13 of the cell trapping device 101 via the first valve 221 and the first pump 211 .

In the first space 11, the target cells 151 adsorbed to the cell adsorption layer 30 are retained in the culture medium. Excess culture medium passes through the filtration part 22 and the second space 12 and is discharged from the discharge port 14 . Further, it reaches the second recovery chamber 232 via the second pump 212 and the second valve 222 and is held therein. This makes it possible to create an environment in which the target cells 151 are held in the culture medium.

As described above, according to the present embodiment, by adsorbing the target cells in the sample liquid to the cell adsorption layer 30, the target cells are separated from the sample liquid and furthermore, contaminants are removed to purify the target cells. can be retained in the medium. Therefore, it is possible to culture the target cells in the medium, or use the separated target cells for inspection, analysis, and the like.

In this embodiment, the cell trapping device 101 includes a first pump 211 and a second pump 212 that are driven bidirectionally. If the outlet 14 and the filter section 22 are sealed except for the outlet 14 and the filter section 22, one of the pumps may be omitted. Alternatively, the first pump 211 is drivable in the forward direction and does not drive in the reverse direction but allows fluid to pass through, and the second pump 212 is drivable in the reverse direction and drives in the forward direction. It may be a one-way pump (that is, without a non-return valve) that does not allow fluid to pass through.

Moreover, the cell adsorption layer 30 preferably contains intermediate water in advance before being brought into contact with the test liquid. For this purpose, a first valve 221 connects the medium holding chamber 214 and the first pump 211 and a second valve connects the second pump 212 and the second collection chamber before introducing the test liquid into the first space 11 . 232, the first pump 211 and the second pump 212 may be driven forward. As a result, the medium flows out of the medium holding chamber 214 and is introduced into the first space 11 from the inlet 13 of the cell trapping device 101 via the first valve 221 and the first pump 211, and the cell adsorption layer 30 contains intermediate water. will come to

The cell-trapping device, cell-trapping apparatus, and cell-trapping method of the present disclosure can be applied to blood-circulating tumor cells, stem cells, vascular endothelial cells, nerve cells, dendritic cells, smooth muscle cells, and fibers contained in intravascular fluids such as blood and lymph. Cells such as blast cells, cardiomyocytes, skeletal muscle cells, hepatic parenchymal cells, hepatic non-parenchymal cells, pancreatic islet cells and the like can be favorably trapped, and are favorably used in various medical treatments.

10, 60 Upper plates 10a, 40a, 60a, 70a, 90a Upper surfaces 10b, 40b, 60b, 70b, 90b Lower surfaces 10r1, 10r2 Recess 11 First space 12 Second space 13 Inlet 14 Outlet 20 Spacer 21 Openings 21c, 21d , 21e region 22 filtration part 30, 80 cell adsorption layers 40, 90 lower plate 60r, 90r recess 70 filter 70h, 80h through hole 101, 102 cell trapping device 103 cell trapping device 151 target cell 152 blood cell component 153 protein 201 holding chamber 202 Pump 211 First pump 212 Second pump 213 Test liquid holding chamber 214 Medium holding chamber 215 Cleaning liquid holding chamber 216 Control circuit 221 First valve 222 Second valve 231 First collection chamber 232 Second collection chamber

Claims (21)

a first space;
a second space;
an inlet connected to the first space;
an outlet connected to the second space;
a filtration unit that connects the first space and the second space and suppresses passage of particles larger than a predetermined size;
a cell adsorption layer arranged in contact with at least the first space and adsorbing target cells in the test solution;
A cell trapping device, comprising: 2. The cell trapping device according to claim 1, wherein at least one of said first space and said second space is closed except for said inlet, said outlet, and said filtering section. 3. The apparatus according to claim 1 or 2, further comprising an upper plate and a lower plate spaced apart from said upper plate by a predetermined gap, wherein said filtering section is formed by the gap between said upper plate and said lower plate. A cell trapping device as described. 4. The cell trapping device according to claim 3, wherein the cell adsorption layer is arranged on the lower plate. 4. The cell-trapping device of Claim 3, wherein said gap is a capillary space. 6. The cell trapping device according to claim 5, wherein said gap is 30 [mu]m or less. further comprising a plate-shaped spacer having an opening disposed between the upper plate and the lower plate;
The filtering part is formed in the opening of the spacer,
the top plate has an upper surface and a lower surface;
the inlet and the outlet are formed in the upper plate in contact with the upper surface;
The first space and the second space are formed in the upper plate in contact with the lower surface,
4. The cell trapping device of claim 3, wherein at least part of said first space and at least part of said second space are in contact with said opening of said spacer. The cell trapping device according to any one of claims 1 to 7, wherein the cell adsorption layer is arranged only in the first space. The cell trapping device according to any one of claims 3 to 7, wherein the cell adsorption layer is arranged on the entire upper surface of the lower plate. 3. The cell trapping device according to claim 1, further comprising a plate-like filter having a plurality of through-holes, wherein said filtering section is said plurality of through-holes. 10. The cell trapping device according to claim 9, wherein the plurality of through-holes have a diameter of 30 [mu]m or less. 11. The cell trapping device according to claim 10, wherein said cell adsorption layer is positioned around said plurality of through-holes of said filter and is in contact with said first space. further comprising a top plate having a top surface and a bottom surface and a bottom plate having a top surface and a bottom surface;
The introduction port is formed in the upper plate in contact with the upper surface,
The first space is formed in the upper plate in contact with the lower surface,
the outlet is formed in the lower plate in contact with the lower surface;
the second space is formed in the lower plate in contact with the upper surface;
13. The cell trapping device of claim 12, wherein the filter is positioned between the lower surface of the upper plate and the upper surface of the lower plate. The target cells include circulating tumor cells, stem cells, vascular endothelial cells, nerve cells, dendritic cells, smooth muscle cells, fibroblasts, cardiomyocytes, skeletal muscle cells, hepatic parenchymal cells, non-hepatic non-parenchymal cells, and pancreatic cells. 14. The cell trapping device according to any one of claims 1 to 13, comprising at least one selected from the group consisting of islet cells, wherein said cell adsorption layer adsorbs said target cells. the cell adsorption layer comprises a first biocompatible polymeric material,
The first biocompatible polymeric material is any one or more of the polymers represented by the following formulas (1) to (4), or a monomer constituting the polymer represented by the formula (1) , polyethylene glycol, polyvinyl pyrrolidone, polyvinyl methyl ether, polymethoxyethyl acrylamide, polymethoxyethyl methacrylamide, polymethoxyethyl vinyl ether, polytetrahydrofurfuryl acrylate and polytetrahydrofurfuryl methacrylate. 15. A cell-trapping device according to any one of claims 1 to 14, comprising a copolymer with a monomer constituting

(In formula (1), R ¹ is a hydrogen atom or a methyl group, R ² is a methyl group or an ethyl group, m is an integer of 1 or more and 3 or less, and n is a repeating unit.)

(In Formula (2), R1 is a hydrogen atom or a methyl group, and n is a repeating unit.)

(In formula (3), X and Y are each independently a hydrogen atom, an alkali metal atom, or an ammonium group, m is an integer of 0 or more and n is a repeating unit.)

(In formula (4), R ¹ is a hydrogen atom or an organic group having 1 to 30 carbon atoms, and n is a repeating unit.) a cell trapping device according to any one of claims 1 to 15;
a pump connected to at least one of the inlet or the outlet of the cell trapping device;
A cell trapping device. 17. The cell trapping device of claim 16, wherein said pump is a bi-directional pump. a test liquid holding chamber that holds the test liquid;
a medium holding chamber for holding medium;
a first collection chamber holding a waste liquid;
a second collection chamber for holding waste fluid;
a cleaning solution holding chamber for holding a cleaning solution;
a first valve selectively connecting one of the test solution holding chamber, the medium holding chamber and the first collection chamber to the introduction port;
a second valve selectively connecting one of the second collection chamber and the wash solution holding chamber to the outlet;
18. The cell trapping device of claim 16 or 17, further comprising: A cell capturing method for separating target cells from a test solution,
preparing the cell trapping device according to claim 1,
Adsorbing the target cells to the cell adsorption layer by introducing a test solution from the inlet and discharging it from the outlet;
Cell capture method. The test solution contains other cells,
holding the other cell in the first space in the adsorbing step;
20. The method according to claim 19, wherein after the adsorption step, another liquid is introduced from the discharge port and discharged from the introduction port, thereby discharging the other liquid held in the first space from the introduction port. A cell capture method as described. A cell capturing method for separating target cells from a test solution,
preparing the cell trapping device according to claim 18,
introducing the test liquid held in the test liquid holding chamber from the introduction port, discharging it from the discharge port, and holding it in the second recovery chamber;
introducing the cleaning liquid held in the cleaning liquid holding chamber from the discharge port, discharging it from the introduction port, and holding it in the first collection chamber;
The medium held in the medium holding chamber is introduced from the inlet, discharged from the outlet, and held in the second collection chamber.
Cell capture method.

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