WO2020250844A1 - Method for separating cells - Google Patents

Abstract

A method for separating cells, said method comprising: without putting a blood sample containing target cells in an environment at 4°C, feeding the blood sample to a cell separation device, which comprises a separation area provided with a microchannel, a sample introduction port and a discharge port, from the sample introduction port to thereby introduce the blood sample into the cell separation device; separating the target cells from the blood sample in a continuous flow of a liquid which contains the blood sample; and then collecting the thus separated target cells from the discharge port.

Description

How to isolate cells

The present invention relates to a method for separating target cells.
The present application claims priority with respect to Japanese Patent Application No. 2019-11504 filed in Japan on June 14, 2019, the contents of which are incorporated herein by reference.

Separating specific cells from a humoral mixed sample (eg blood or culture medium) is a technique required for basic research, diagnosis and treatment. As a method for separating cells, a method for separating cells by a microchannel is known. As a method for separating cells by a microchannel, a method having a plurality of different channel widths and separating cells according to the channel width (for example, Patent Document 1) and an antibody fixed to the microchannel are used. A method for capturing cells (for example, Patent Document 2) is known. Further, as described in Patent Documents 3 and 4 and Non-Patent Document 1, there is also a method of separating cells according to their size using a fluid device using a principle called a deterministic lateral displacement (DLD). Are known.

When the sample containing the target cells to be separated is a blood sample, the blood is usually refrigerated at a temperature of about 4 ° C. before the cell separation treatment.

JP-A-2018-158328 Japanese Unexamined Patent Publication No. 2013-29391 International Publication No. 2016/136273 International Publication No. 2011/11174 JP-A-2017-184685

However, in the method of separating target cells by a microchannel, the present inventors, when the sample is a blood sample, coagulates blood in the channel when a blood sample collected from a living body is introduced into the microchannel. It was found that this occurred and the flow path was clogged.
Therefore, the present invention is a method for separating target cells by a microchannel, in which when the blood sample is introduced into the microchannel, the target cells in the blood sample are separated without coagulation of blood in the channel. The purpose is to provide a way to do this.

In order to achieve the above object, the present invention has the following configurations.
[1] Without placing the blood sample containing the target cells in an environment of 4 ° C. or lower, the blood sample is added from the sample introduction port to a cell separation device having a separation area having a microchannel, a sample introduction port and a discharge port. And to introduce the blood sample into the cell separation device.
Separation of the target cells from the blood sample in a continuous stream of liquid containing the blood sample.
Collecting the separated target cells from the outlet and
A method for separating cells including.
[2] The method for separating cells according to [1], wherein not placing in an environment of 4 ° C. or lower is placing in an environment of 10 ° C. or higher and 40 ° C. or lower.
[3] The method for separating cells according to [1] or [2], further comprising adding and flowing a buffer solution from the buffer inlet of the cell separation device.
[4] The method for separating cells according to [3], wherein the temperature of the buffer solution is 10 ° C. or higher and 40 ° C. or lower.
[5] The method for separating cells according to any one of [1] to [4], wherein the blood sample is blood diluted with a diluent.
[6] The method for separating cells according to any one of [1] to [5], wherein the separation area having the microchannel is a separation area having a deterministic transverse substitution method microchannel structure.
[7] The blood sample further contains non-target cells and contains
Prior to introduction into the cell separation device, a target capture substance that recognizes a characteristic structure existing on the surface of the target cell is added to the blood sample, and a complex of the target cell and the target capture substance is added. Including generating
When the target cells are separated, cells having a size smaller than the threshold move with the flow of the blood sample, and the complex having a size equal to or larger than the threshold is displaced obliquely with respect to the flow. It is to separate by moving,
The method for separating cells according to any one of [1] to [6], wherein the target cells are collected as the complex in the collection.
[8] The blood sample further contains non-target cells and contains
Prior to introduction into the cell separation device, a target capture substance that recognizes a characteristic structure existing on the surface of the target cell is added to the blood sample, and a composite of the target cell and the target capture substance is added. Including producing the body
When the target cells are separated, cells having a size smaller than the threshold move with the flow of the blood sample, and the complex having a size equal to or larger than the threshold moves obliquely with respect to the flow. The method for separating cells according to any one of [1] to [6], wherein the cells are separated by the above-mentioned method.
[9] The target capture substance comprises a conjugate of a target capture molecule having a characteristic structure existing on the surface of the target cell or a non-target cell and a substance carrying the target capture molecule [7]. ] Or the method for separating cells according to [8].
[10] The method for separating cells according to [9], wherein the target capture molecule is an antibody, a peptide aptamer, a lectin, an intercellular adhesion molecule, a sugar chain, or a cell-recognizable polymer.
[11] The method for separating cells according to [9] or [10], wherein the substance supporting the target capture molecule is polystyrene or latex.

According to the present invention, in the method of separating target cells by a microchannel, when a blood sample is introduced into the microchannel, the target cells can be separated without coagulation of blood in the channel.

It is a schematic diagram explaining the basic principle of the separation method which concerns on embodiment of this invention. It is a schematic diagram explaining the separation device provided with the basic structure (separation area) of the DLD microchannel which concerns on embodiment of this invention. FIG. 5 is a schematic vertical cross-sectional view of a separation device provided with a basic structure of a DLD microchannel according to an embodiment of the present invention. It is a schematic diagram explaining the separation apparatus provided with the liquid feeding part and the collecting part which concerns on embodiment of this invention. It is a schematic diagram explaining the cell suspension containing the cell and the target capture substance which concerns on embodiment of this invention. It is a schematic diagram explaining the separation method which captures the target cell which concerns on embodiment of this invention. It is a schematic diagram explaining the separation method which captures the non-objective cell which concerns on embodiment of this invention. It is a schematic diagram explaining the method of removing the microaggregate which concerns on embodiment of this invention. It is a schematic diagram explaining the pillar structure of the microaggregate removing device which concerns on embodiment of this invention. It is a schematic diagram explaining the microaggregate removing device (a mode having two microchannel structures) which concerns on embodiment of this invention. It is a top view which shows an example of an integrated cell separation device. It is a side view which shows an example of an integrated cell separation device. It is a plan view of an integrated cell separation device, an enlarged view of a portion where the flow width is converged, and an enlarged view of a cell separation portion. It is a figure which shows the blood flow of the blood separation device when the blood sample stored at 25 degreeC for 4 hours or 24 hours after blood collection was used. It is a figure which shows the blood flow of the blood separation device immediately after the introduction of a blood sample when the blood sample stored at 4 degreeC for 4 hours after blood collection is used. It is a figure which shows the blood flow of the blood separation device 40 minutes after the introduction of a blood sample when the blood sample stored at 4 degreeC for 4 hours after blood collection is used.

1. 1. Principle of separating cells One aspect of the present invention is a separation area and sample having a microchannel (hereinafter, also referred to as a microchannel) without placing a blood sample containing a target cell in an environment of 4 ° C or lower. The blood sample is added to a cell separation device provided with an inlet and an outlet from the sample inlet and flowed, the blood sample is introduced into the cell separation device, and a liquid containing the blood sample is continuously flowing. The present invention relates to a method for separating cells, which comprises separating the target cells from the blood sample and collecting the separated target cells from the outlet.

By introducing the blood sample into a cell separation device having a separation area having a microchannel without placing it in an environment of 4 ° C. or lower, the blood sample is not aggregated in the microchannel and the purpose in the blood sample. Cells can be isolated. After collecting a blood sample from a living body and before introducing it into a cell separation device, the blood sample is usually stored refrigerated at about 4 ° C. However, it is surprising that agglutination in the microchannel was suppressed by not placing the blood sample in an environment below 4 ° C. When examining the target cells after separating the target cells from the blood sample, it may be desired that the anticoagulant reagent is not mixed. According to one aspect of the present invention, the target cells can be separated using the cell separation device without adding an anticoagulation reagent to the blood sample. Therefore, the isolated target cells can be used for various tests.

"Do not place the blood sample in an environment of 4 ° C or lower" means that the blood sample is not placed in an environment of 4 ° C or lower for a certain period of time. That is, "do not place the blood sample in an environment of 4 ° C or lower" means that the blood sample is not stored in an environment of 4 ° C or lower. Specifically, it is preferable not to leave the blood sample at 4 ° C. or lower for 1 hour or longer, more preferably not to leave it at 4 ° C. or lower for 3 hours or longer, and particularly preferably not to leave it at 4 ° C. or lower for 8 hours or longer. For example, after collecting blood, the blood sample is preferably 10 ° C. or higher and 40 ° C. or lower, more preferably 15 ° C. or higher and 40 ° C. or lower, further preferably 20 ° C. or higher and 40 ° C. or lower, and particularly preferably 25 ° C. or higher and 37 ° C. or lower. It is preferably introduced into the cell separation device within 3 days (ie, within 72 hours), more preferably within 2 days (ie, within 48 hours), and particularly preferably within 1 day (ie, within 24 hours).

The lower limit of the time from blood collection to introduction into the cell separation device is not particularly limited. When the collected blood is used as it is as a blood sample, the blood sample may be introduced into the cell separation device immediately after the blood collection. If the blood sample is a mixture of blood and a diluent described below, the blood sample may be immediately introduced into the cell separation device after mixing the blood with the diluent.

As the blood sample, the blood sample may be introduced into the cell separation device within 1 hour or more and 3 days, 3 hours or more and 2 days, or 6 hours or more and 1 day after blood collection.

The temperature at which the blood sample is placed and the range of the period can be arbitrarily combined. For example, the blood sample may be introduced into a cell separation device after blood collection, storage at 10 ° C. or higher and 40 ° C. or lower for a period of 1 hour or more and 3 days or less. The blood sample may be stored in a cell separation device at 15 ° C. or higher and 40 ° C. or lower for 3 hours or more and 2 days or less after blood collection. After collecting blood, the blood sample may be stored at 20 ° C. or higher and 40 ° C. or lower for a period of 6 hours or more and 1 day or less, and then introduced into a cell separation device. The blood sample may be introduced into a cell separation device after being stored at 25 ° C. or higher and 37 ° C. or lower for a period of 6 hours or more and 1 day or less after blood collection.

Introducing into a cell separation device may include adding a buffer solution from the buffer introduction port of the cell separation device and flowing it. The buffer solution to be used can be appropriately selected and used according to at least one of the target blood sample, the target cells to be separated, and the contained microaggregates. As the buffer solution, one or a combination of a plurality of isotonic solutions can be used in order to avoid the influence on the cells. For example, physiological saline or PBS may be used as the buffer solution. In this case, the temperature of the buffer solution introduced from the buffer introduction port is a temperature exceeding 4 ° C., preferably 10 ° C. or higher and 40 ° C. or lower, preferably 15 ° C. or higher and 40 ° C. or lower, and more preferably 20 ° C. or higher and 40 ° C. or lower. Particularly preferably, it is 25 ° C. or higher and 37 ° C. or lower.

Further, as the blood sample, the blood may be used as it is, or the blood may be diluted with an arbitrary diluent and used. Examples of the diluent include the above-mentioned buffer solution and the like. When diluting with a diluent, the temperature of the diluent is a temperature exceeding 4 ° C, preferably 10 ° C or higher and 40 ° C or lower, more preferably 15 ° C or higher and 40 ° C or lower, and further preferably 20 ° C or higher and 40 ° C or lower. Particularly preferably, it is 25 ° C. or higher and 37 ° C. or lower.

The cell separation device having a separation area having a microchannel structure is not particularly limited, but a cell separation device having a separation area having a deterministic Lateral Displacement (DLD) microchannel structure is preferable.
FIG. 1 is a schematic diagram illustrating the basic principle of the deterministic transverse substitution method in the embodiment of the present invention. Hereinafter, DLD will be described.

1-1. Deterministic transverse substitution (DLD)
In the deterministic transverse displacement method, when a dispersion of particles is flowed through a slightly displaced pillar structure, large particles flow diagonally from the change in flow that occurs around the pillars, whereas small particles ride on laminar flow. This is a method of realizing sorting by size by utilizing the property of linearly advancing globally (see Non-Patent Document 1 and FIG. 1). The deterministic transverse substitution method is a method using a flow path in which regular obstacles (hereinafter sometimes referred to as micropillars or microposts) are arranged while being displaced. In the deterministic transverse substitution method, small particles travel straight along the flow, while large particles travel diagonally along the displacement of the obstacle as the flow changes around the obstacle.

In general, this DLD principle is also applied to the separation of cells, for example, the separation of components in blood, specifically erythrocytes, leukocytes, and circulating tumor cells (hereinafter sometimes referred to as CTC). There is. By flowing blood cells on the dimensional selection micropost structure and passing them through a microfluidic device using DLD, relatively large cells can be selected by the DLD microchannel depending on the size of the cells. However, when the size of target cells and non-target cells present in blood are similar, it may be difficult to separate them. Examples of cell combinations in difficult cases include cases where the target cell is a tumor cell and the non-target cell contains leukocytes.

In the method for separating cells of the present embodiment, as described later, a target capturing substance that recognizes a characteristic structure existing on the surface of a target cell or a non-target cell may be used. Specifically, by adding the target capture substance to the cell suspension to generate a complex of the target cell or the non-target cell and the target capture substance, the size is increased between the target cell and the non-target cell. Make a difference. Then, the target cells can be separated by using a cell separation device having a basic structure of the DLD microchannel.

1-2. Diagonally displaced cell diameter threshold in the DLD principle, cell separation device Based on the DLD principle described in Non-Patent Document 1, the threshold of the obliquely displaced cell diameter depending on the diameter of the target cell to be separated. (Dc) can be set. More specifically, the setting of the threshold value Dc can be obtained from the following equation (1).
Dc = 2ηGε ・ ・ ・ (1)
Dc: Threshold for the diameter of cells displaced in the oblique direction η: Variable G: Gap between pillars
ε: Pillar shift angle (tanθ)

Then, by solving the above equation (1), the following approximate equation (2) is obtained.
Dc = 1.4Gε ^0.48 ... (2)

Then, from the rule of thumb, since the separation of the target cells is good when 0.06 <ε <0.1, the following relational expression (ε = tanθ = 1/15 = 0.067) is used. 3) can be derived.
G≈2.62057Dc ... (3)

Then, from the above formula (3), the inter-pillar gap G is calculated based on the threshold Dc of the diameter of the cells displaced in the oblique direction. Then, a cell separation device having a basic structural portion in which a pillar group having a gap G between pillars (hereinafter, may be referred to as an obstacle structure) is installed can be produced. In this way, a cell separation device having a DLD microchannel having a target threshold Dc can be prepared.

In the present specification, the diameter of a cell is the diameter of the sphere when the cell is a sphere. When a cell is not a sphere, a sphere having the same volume as the cell is assumed, and the diameter of the sphere is defined as the diameter of the cell. Further, in the present specification, the diameter of a cell may be referred to as a cell size or a cell size.

When separating the complex of the target cell and the target capture substance, the diameter of the complex is used as the diameter of the target cell. The diameter of the complex is defined as the sum of the diameter of the target capture substance and the diameter of the target cell. The diameter of the target capture substance is the diameter of the sphere when the target capture substance is a sphere. When the target trapping substance is not a sphere, a sphere having the same volume as the target capturing substance is assumed, and the diameter of the sphere is defined as the diameter of the target capturing substance.

First, FIG. 1 shows the basic structure 20 of the DLD microchannel (hereinafter, may be referred to as a separation area). Obstacle structures 21 arranged diagonally according to a certain rule with respect to the flow direction of the fluid in the direction of the arrow are provided. In the fluid directed in the direction of the arrow, the flow velocity changes around the obstacle structure 21. Utilizing the change in flow velocity due to the continuously arranged obstacle structure 21, when the diameter of the cell (referred to as a particle in the description of FIG. 1) contained in the fluid is equal to or larger than the threshold value, the particle of the particle is used. The direction of travel can be changed.

Regarding the change in the traveling direction, the particles 22 having a diameter equal to or larger than the threshold value are displaced in the oblique direction according to the arrangement of the obstacle structures 21 arranged continuously. On the other hand, the particles 23 having a diameter less than the threshold value do not follow the behavior of the particles 22 described above, and go straight along the flow direction while bypassing the obstacle structure 21.

In this way, by designing the arrangement pattern of the obstacle structure 21 in which the threshold value is set so that the particles of the target size can be separated according to a known method (for example, the method described in Non-Patent Document 1). Depending on the size of the particles contained in the fluid, the particles can be separated in a direction orthogonal to the flow direction. Furthermore, by providing different recovery channels in the downstream portion in the flow direction, it is possible to recover the separated particles.

The shape of the obstacle structure 21 is not limited to the cylindrical structure as shown in FIG. The obstacle structure 21 can have a polygonal prism structure such as a triangular prism as long as it has a shape capable of causing a desired change in flow velocity.

FIG. 2 is a schematic diagram illustrating a cell separation device 30 having a basic structure of a DLD microchannel according to an embodiment of the present invention. Hereinafter, the basic structure of the cell separation device 30 will be described with reference to a top view. First, as a fluid inlet structure, a sample introduction port 31 and a buffer introduction port 32 are provided.

Further, the fluid outlet structure includes a first discharge port 33 and a second discharge port 34. The first discharge port 33 is a fraction containing particles having a size equal to or larger than a threshold value (that is, a certain size or more), which is displaced in a direction according to the arrangement of diagonally arranged obstacle structures. Displace. The second discharge port 34 discharges a fraction containing particles having a size smaller than the threshold value (that is, less than a certain size) traveling straight along the flow direction. The number of the first discharge port 33 and the second discharge port 34 is not limited to one as shown in FIG. 2, and may have a plurality of outlets depending on the purpose.

The basic structure 20 of the DLD microchannel having a micropillar structure is continuous between the sample introduction port 31 and the buffer introduction port 32 of the cell separation device and the first discharge port 33 and the second discharge port 34. Is located in. Due to the region of the continuously arranged basic structure 20, cells contained in the blood sample can be separated in a direction orthogonal to the flow direction of the blood sample depending on the size thereof. Regarding the portion where the basic structure 20 is continuously arranged, the basic structure of the same design may be continuous in all the areas, and the area where the design is gradually changed may be set according to the purpose. It is possible. The design of the basic structure 20 is an arrangement pattern of the basic structure 20 when the cell separation device 30 is viewed from above.

Examples of the buffer solution introduced from the buffer introduction port 32 include the above-mentioned buffer solution.

Regarding the structure of the inlet portion from the sample introduction port 31 to the inside of the fluid device 30, it is preferable to provide a partition structure 45 having a constant width as shown in FIG. 2 because it is necessary to flow a blood sample as described later. The mixed solution sent from the sample introduction port 31 flows while maintaining a laminar flow (that is, a flow forming a layer parallel to the flow direction) with the buffer solution sent from the buffer introduction port 32. Cells having a size equal to or larger than the threshold value are separated from the layer of the mixed solution by being displaced diagonally with respect to the flow direction according to the above-mentioned separation principle. On the other hand, cells having a size less than a certain threshold flow straight along the flow of the blood sample layer. As described above, for the purpose of separating cells, the position of the sample introduction port 31 needs to be set closer to the second discharge port 34 side of the fluid device 30. That is, the position of the sample introduction port 31 needs to be installed closer to the second discharge port 34 than to the first discharge port 33.

Further, the structure of the first discharge port 33 and the second discharge port 34 can be appropriately changed in design according to the purpose in order to adjust the amount of the discharged liquid. For example, a partition wall 38 near the discharge port is provided so that the ratio of the dimension of the upper portion y and the dimension of the lower portion x when the cell separation device 30 is viewed from the upper surface of FIG. 2 is 1: 1. In this case, the ratio of the amount of liquid obtained from each outlet is approximately 1: 1. On the other hand, when the partition wall 38 near the discharge port is provided so that the ratio of the dimension of the upper portion y to the dimension of the lower portion x is 49: 1, the amount of liquid obtained from the first discharge port 33 is. It will be 1/50. As a result, it is possible to concentrate the concentration of cells having a size equal to or larger than the threshold value of the liquid obtained from the first outlet 33 approximately 50 times. As described above, the cell separation device 30 can be used not only for separating cells in a blood sample but also for concentrating target cells depending on the application. As an example, it can be realized by setting the ratio of y and x to 1: 1 or 49: 1, which is shown in the partition wall 38 of FIG. 2 and the branch portion of FIG. 12 described later.

FIG. 3 is a schematic view of a vertical cross section of the cell separation device according to the embodiment of the present invention. In the cell separation device 30, the flow path structure portion 36 and the planar structure portion 37 are joined to each other. The cell separation device 30 constitutes the basic structure 20 of the DLD microchannel. The flow path structure portion 36 has shapes such as a sample introduction port 31, a buffer introduction port 32, a first discharge port 33, and a second discharge port 34. The space between the flow path structure portion 36 and the planar structure portion 37 is the flow path space 35. Further, the sample introduction port 31, the buffer introduction port 32, the first discharge port 33, and the second discharge port 34 are changed by appropriately joining tubes and providing joints with a syringe or the like. Can also be used in addition to.

The height of the flow path space 35 is not limited as long as it is set to a height at which cells or complexes having a size equal to or larger than the threshold value can pass through. The height of the flow path space 35 is such that two or more cells or complexes having a size equal to or larger than the threshold value cannot exist at the same time in the height direction of the flow path space 35 in order to perform accurate cell separation. It is desirable to set it to.

The member of the flow path structure portion 36 and the method for manufacturing the flow path structure portion 36 can be manufactured by appropriately selecting any known method. As the material of the member, for example, glass, silicone, dimethylpolysiloxane, plastic or the like can be used. The planar structure portion 37 is not particularly limited as long as it is a flat material that can be bonded to the flow path structure portion 36, but it is preferable to use glass, plastic, or the like having strength.

The cell separation device 30 has a basic structure, a sample introduction port, a buffer introduction port, a first discharge port, and a second discharge port, respectively, and there is no problem as long as the flow path space is formed. For example, the member on the side of the planar structure portion 37 shown in FIG. 3 may have at least one of the shapes of a basic structure, a sample introduction port, a buffer introduction port, a first discharge port, and a second discharge port. it can. For example, the planar structure portion 37 may have a basic structure, and the flow path structure portion 36 may include a sample introduction port, a buffer introduction port, a first discharge port, and a second discharge port. Further, for example, the flow path structure portion 36 may include a basic structure, a sample introduction port, a buffer introduction port, a first discharge port, and a second discharge port.

FIG. 4 is a schematic view illustrating a separation device including a liquid feeding unit and a collecting unit according to an embodiment of the present invention. The separation device 40 has a cell separation device 30 having a basic structure of a DLD microchannel. The cell separation device 30 includes a sample liquid supply unit 41 in the sample introduction port 31 and a buffer liquid supply unit 42 in the buffer introduction port 32, respectively. Further, the first discharge port 33 is provided with the first collection unit 43, and the second discharge port 34 is provided with the second collection unit 44.

The sample liquid feeding unit 41 and the buffer liquid feeding unit 42 each have a mechanism provided with a liquid feeding system, and can independently feed the liquid at a constant speed. These are not particularly limited as long as they are mechanisms capable of delivering liquid at a constant speed, but for example, liquid feeding by a syringe pump or the like is preferable. Further, if necessary, the sample liquid feeding unit 41 may be provided with a stirring mechanism for preventing precipitation and aggregation of the complex 5 and an automation mechanism for adjusting the mixed liquid (also referred to as blood sample) 4.

The first recovery unit 43 and the second collection unit 44 are not particularly limited as long as they have a mechanism that can collect the discharged liquid, but even if they are provided with a mechanism that can fractionate the discharged liquid immediately before or after the start of separation. good. For example, a blood sample may be introduced from the sample introduction port 31 after the buffer solution is sent from the sample introduction port 31. In such a case, the first recovery unit 43 and the second collection unit 44 may be provided with a mechanism capable of fractionating the discharged liquid over time. As a result, the fraction containing only the buffer solution before the blood sample is sent and the fraction containing the blood sample can be collected separately.

2. 2. Cell Separation Method Hereinafter, the separation method according to one aspect of the present invention will be described with reference to the drawings.

The present invention is, as described above, a method of separating target cells from a blood sample in a continuous stream of liquid. The "blood sample" is not particularly limited as long as it is a blood sample containing target cells. Examples of blood samples include whole blood, serum, plasma, and the like. Further, the blood sample may be whole blood, serum, plasma or the like diluted with a diluent.
In the method for separating cells according to the embodiment of the present invention, the blood sample may contain non-target cells in addition to the target cells. In that case, the cell separation method is to add a target capture substance that recognizes the characteristic structure existing on the surface of the target cell to the target cell and the target cell before introducing the blood sample into the cell separation device. It may include producing a complex with a trapping agent and separating the complex using a cell separation device.
The complex of the target cell and the target capture substance is preferably produced at a temperature of more than 4 ° C, more preferably 10 ° C or higher and 40 ° C or lower, for example, 15 ° C or higher and 40 ° C or lower, 20 ° C or higher and 40 ° C. ° C or lower, or 25 ° C or higher and 37 ° C or lower.

2-1. Step of Creating a Complex of Target Cell and Target Captured Substance The cell separation method according to the embodiment of the present invention may include forming a complex of target cell or non-target cell and target capture substance. FIG. 5 is a schematic diagram illustrating a blood sample containing cells and a target capture substance in the method for separating cells according to the embodiment of the present invention. First, the target capture substance 12 which is a conjugate of the target capture molecule 10 that recognizes the characteristic structure existing on the surface of the target cell and the substance 11 that supports the target capture molecule 10 is used. The target capture substance 12 is mixed in the cell suspension 1 with respect to the blood sample 1 containing the target cells 2 and the non-target cells 3. As a result, in the obtained mixed solution 4, a complex 5 of the target cell 2 and the target capturing substance 12 is formed.

The non-target cells 3 contained in the blood sample 1 are not limited to one type. The type and number of the non-target cells 3 are not limited as long as they do not have the characteristic structure existing on the surface of the target cells recognized by the target capture molecule 10. Further, the target capture molecule 10 is a molecule that recognizes the characteristic structure existing on the surface of the target cell 2 and does not recognize the characteristic structure existing on the surface of cells other than the target cell 2. It is not limited to one type, and a plurality of necessary types and numbers can be selected and used as appropriate.

More specifically, the target capture substance 12 may be prepared by supporting a plurality of target capture molecules 10 of the same or different types on the substance 11 that supports the target capture molecule 10. Further, the same type or different types of target trapping molecules 10 may be supported on the substance 11 that supports a plurality of target capturing molecules 10, and the target capturing substance 12 may be appropriately selected according to the intended use. Can be created. In order to obtain the target cell with high accuracy, it is preferable to use a plurality of target capture molecules 10 satisfying the above conditions in combination. Further, the size of the substance 11 that supports the target trapping molecule can also be used by using a plurality of different sizes depending on the intended use.

For example, when the target cell 2 further has a, b, ..., And a characteristic structure on the cell surface, the substance 11a having the target capture molecule 10a, the substance 11b having the target capture molecule 10b, ... Can be used respectively. Taking advantage of the fact that the size of the substance 11a carrying the target trapping molecule and the substance 11b supporting the target trapping molecule are different, a plurality of separation threshold areas are passed through in the subsequent separation step, respectively. , B, ..., It is also possible to separate the target cells having the characteristic structures into the respective outlets and collect them.

Further, depending on the intended use, it is also possible to form and separate complex particles of the non-target cell 3 and the target capture molecule to remove the non-target cell.

More specifically, in a blood sample 1 containing the target cell 2 and the non-target cell 3, a conjugate of a target capture molecule 10 that recognizes a structure characteristic of the surface of the target cell and a substance 11 that carries the target capture molecule 10. The target capture substance 12 is added. As a result, it is possible to form a complex 5 of the non-target cell 3 and the target capture substance 12 in the mixed solution 4 (not shown).

Similarly, in this case as well, the target capture molecule 10 is a molecule that recognizes the characteristic structure existing on the surface of the non-target cell 3 and does not recognize the characteristic structure existing on the surface of the target cell 2. If it is a molecule, it is not limited to one type, and a plurality of necessary types and numbers can be selected and used as appropriate.

The target capture molecule 10 is a molecule that recognizes the characteristic structure existing on the surface of the cell forming the complex 5, and is a molecule that does not recognize the characteristic structure existing on the surface of other cells. , There is no particular limitation. As the target capture molecule 10, for example, any one of an antibody, a peptide aptamer, a lectin, an intercellular adhesion molecule, a sugar chain, and other cell-recognizable macromolecules, or a plurality of these may be appropriately used. it can.

As the substance 11 that supports the target capture molecule, a member formed of a polymer such as a metal, an inorganic material, or a resin can be used. This member may be formed from a single material, or may be used in combination of a plurality of types. When a plurality of types of materials are used, it is preferable to use a member formed so that each component is uniformly kneaded. Further, a high molecular polymer may be selected as the material of the substance 11 that supports the target trapping molecule, and specifically, polystyrene, latex, or the like can be used.

The member of the substance 11 that supports the target trapping molecule is not particularly limited as long as it is made of the above-mentioned material. Since it will be an important factor in the subsequent separation stage, it is preferable to use a substance 11 supporting the target capture molecule having a uniform specific gravity. Furthermore, when the specific gravity of the substance 11 supporting the target capture molecule is small during the reaction in the blood sample 1, the dispersion in the blood sample may not be uniform, so that the specific gravity is close to or higher than that of water. It is preferable to select a member having a slightly larger size. The specific gravity of the material 11 which carries the target capture molecules ^may be 1.00g / cm 3 ~ 1.20g / cm 3, may be 1.02g / cm 3 ~ 1.12g / cm 3, 1. It may be 04 g / cm ³ to 1.09 g / cm ³ . For example, resin beads having a specific gravity of 1.05 g / cm ³ can be used.

The target capture substance 12 is a conjugate of the target capture molecule 10 and the substance 11 that supports the target capture molecule 10 as described above. The target capture molecule 10 and the substance 11 that supports it can be bound by a known method. For example, in order to immobilize the antibody as the target capture molecule 10 on the substance 11, a method by physical adsorption, a method by chemical bonding, or the like can be used. In the case of chemical bonding, for example, when the surface is made of a material containing a hydroxyl group, the carboxyl group in the antibody is activated esterified, and then the hydroxyl group is reacted with this ester group to immobilize the antibody on the surface. Can be done. It is also possible to use a binding method via Protein A or Protein G. As the target capture substance 12, commercially available substances, for example, beads carrying an antibody can be purchased and used.

When the target capture substance 12 is used, the size of the target capture substance 12 must be selected so that the size of the complex 5 is larger and distinguishable than the size of other cells to be distinguished. is there. That is, it is necessary to select the size of the target trapping substance 12 so that the size of the complex 5 is equal to or larger than the above threshold value. For example, when a complex 5 of the target cell 2 and the target capture substance 12 is formed and the non-target cell 3 is separated, the size of the complex 5 is larger than that of the non-target cell 3 and the above-mentioned threshold value. Must be above.

More specifically, when the diameter of the target cell 2 is 10 to 14 μm and the diameter of the non-target cell 3 is 8 to 12 μm, the diameter of the target capture substance must be at least 2 μm or more. .. For more accurate separation, the diameter of the target trapping substance is preferably 5 μm or more. As long as it does not interfere with the subsequent experiment, it is preferable to use a target trapping substance having a larger diameter. Considering a cell separator based on the DLD principle, a general cell size, and the like, more specifically, the diameter of the target capture substance 12 is 7 μm to 60 μm, preferably 10 μm to 55 μm, and more. It is preferably 15 μm to 50 μm, and more preferably 20 μm to 40 μm. In particular, circulating tumor cells in blood are similar in size to white blood cells, which generally have a diameter of 10 to 20 μm. Therefore, when separating circulating tumor cells from blood, it is preferable to use a target capture substance having a diameter of 20 μm to 40 μm.

In the following description of the cell separation method according to the embodiment of the present invention, the "complex" refers to the above-mentioned complex, but the target cell and the non-target cell are separated without forming the complex. In this case, the invention can be understood by reading "complex" as "target cell".

2-2. Step of separating the complex using a cell separation device The cell separation method according to the embodiment of the present invention is a separation having a microchannel without placing the blood sample containing the complex in an environment of 4 ° C. or lower. A blood sample is added from the sample inlet to a cell separation device provided with an area, a sample inlet and an outlet, and the blood sample is introduced into the cell separation device, and in a continuous flow of liquid containing the blood sample. Includes separating target cells from blood samples. Separating target cells from a blood sample means separating a complex having a size above a determined threshold. Separating the cells of interest from the blood sample allows the blood sample to pass through multiple isolation areas of the cell separation device, and cells with a size smaller than the threshold move with the flow of the blood sample and the threshold. The composite having the above size may include separating by displacing and moving in an oblique direction with respect to the flow.

2-2-1. Step of Creating and Separating a Complex with a Target Cell As a method for separating cells according to the embodiment of the present invention, after creating a complex with a target cell, a cell having a separation area having a DLD microchannel structure. A method of using a separation device to pass a blood sample through multiple separation areas of the cell separation device to separate a complex having a size greater than or equal to a determined threshold can be mentioned.

FIG. 6 is a schematic diagram illustrating a separation method for capturing target cells according to the embodiment of the present invention. In this method, first, the target cell 2 and the target cell 2 and the target cell 2 are mixed with the target capture substance 12 that recognizes the characteristic structure existing on the surface of the target cell with the blood sample containing the target cell 2 and the non-target cell 3. The complex 5 with the trapping substance 12 is formed.

The cell separation device 30 is prepared in a state where the flow path space is filled with a buffer solution in advance to remove air bubbles. To the cell separation device 30, the mixed solution 4 having the non-target cells 3 and the complex 5 is introduced into the cell separation device 30 from the sample introduction port 31, and at the same time, the buffer solution is continuously introduced from the buffer introduction port 32. .. After that, in the cell separation device 30, the non-target cell 3 goes straight by the basic structure 20 (that is, the separation area) having the DLD microchannel structure continuously provided in the cell separation device 30. On the other hand, the complex 5 is displaced obliquely with the flow and moves to be separated from the non-target cell 3. As a result, the complex 5 having the target cells 2 is obtained from the first discharge port 33, and the non-target cells 3 are obtained from the second discharge port 34. As described above, the complex 5 and the non-target cell 3 can be separated.

In the above cell separation method, after forming a complex with a target cell in the first step, a blood sample is passed through a plurality of separation areas of the cell separation device, and the complex has a size equal to or larger than a determined threshold value. It separates the body. At this time, the threshold value depends on the size of the complex, but is suitable from the relationship with the size of the complex, the size of the target capture substance, the target cell, the size of the non-target cell to be distinguished, and the like. Range can be selected.

When the target cell in the blood sample of the present embodiment is a circulating tumor cell, the erythrocyte is 6 to 8 μm, the leukocyte is relatively large, 9 to 15 μm, and the circulating tumor cell is about 10 to 20 μm. is there. Therefore, in order to separate from leukocytes which are non-target cells, the threshold value of the size of the complex is preferably 15 μm or more, more preferably 20 μm or more, further preferably 25 μm or more, and more preferably 30 μm or more. It is more preferable to have. For accurate separation, it is preferable to use a relatively large size target capture material. As described above, the size of the target trapping substance is preferably 7 μm to 60 μm, more preferably 10 μm to 55 μm, further preferably 15 μm to 50 μm, and particularly preferably 20 μm to 40 μm. is there. Therefore, for example, when a target capture substance having a size of 30 μm is used, it can be assumed that the complex with the target cell, the circulating tumor cell, has a size of about 40 to 50 μm. Further, when a target catching substance having a size in a preferable range of 20 μm to 40 μm is used, the size of the complex can be assumed to be 30 μm to 60 μm. The threshold value to be determined by summing up the above is not less than or equal to the size of the non-target cell to be distinguished and less than or equal to the upper limit of the size of the complex assumed from the size of the target cell and the target capture substance. preferable. As a result, the target cells can be separated easily and accurately. Specifically, the threshold value Dc of the size of the complex to be determined is preferably set to 20 to 60 μm, and more preferably set to 30 to 50 μm.

Since the range of this threshold Dc varies depending on the size of the target cell, target cell, non-target cell, and complex, it is determined by appropriately selecting the target cell, non-target cell, and target capture substance. Can be done.

2-2-2. Step of creating and separating a complex with non-target cells In the cell separation method according to the embodiment of the present invention, when the blood sample contains non-target cells in addition to the target cells, blood is added to the cell separation device. Prior to introducing the sample, a target capture substance that recognizes a characteristic structure existing on the surface of the target cell is added to the blood sample to generate a complex of the target cell and the target capture substance. And that the complex may be separated using a cell separation device.
The complex of the non-target cell and the target capture substance is preferably produced at a temperature of more than 4 ° C, more preferably 10 ° C or more and 40 ° C or less, for example, 15 ° C or more and 40 ° C or less, 20. The temperature is 40 ° C or higher and 25 ° C or higher and 37 ° C or lower.

FIG. 7 is a schematic diagram illustrating a separation method for capturing non-target cells according to the embodiment of the present invention. In contrast to the above-mentioned method, in this method, first, a target capturing substance 12 that recognizes a characteristic structure existing on the surface of the non-target cell 3 is mixed with a blood sample containing the target cell 2 and the non-target cell 3. As a result, a complex 5 of the non-target cell 3 and the target capture substance 12 is formed.

Next, as in the above method, the cell separation device 30 is prepared in a state where the flow path space is filled with a buffer solution in advance to remove air bubbles. To the cell separation device 30, the mixed solution 4 containing the target cells 2 and the complex 5 is introduced into the cell separation device 30 from the sample introduction port 31, and at the same time, the buffer solution is continuously introduced from the buffer introduction port 32. .. After that, in the cell separation device 30, the target cells 2 and the complex 5 are separated in the flow direction and the vertical direction by the basic structural portion 20 continuously provided in the cell separation device 30. As a result, the complex 5 having the non-target cells 3 is obtained from the first discharge port 33, and the target cells 2 are obtained from the second discharge port 34. As described above, cells can be separated.

The above two methods can be used properly according to the purpose. For example, when the target capture molecule 10 with respect to the target cell 2 is clear and highly specific, the method described in FIG. 6 can be preferably used. On the other hand, when the target capture molecule 10 for the target cell 2 is not clear, the target cell 2 can be separated by using the method described in FIG. 7. In addition, each method can be appropriately used according to the type and number of non-target cells 3.

Furthermore, the above two methods can be used properly according to the purpose after separation. For example, when the target cell 2 is to be recovered as it is without forming the complex 5, it can be carried out by the method described with reference to FIG. 7. Also, when the method described in FIG. 6 is carried out, the complex 5 is dissociated using any known method, the target cell 2 and the target capture substance 12 are separated, and further, any known method is used. Although it is possible to take a method of separating only the target cells 2 using the above, in this case, it is preferable to carry out by the method described with reference to FIG. 7 because the procedure is complicated.

2-3. Step of Recovery The method for separating cells according to one aspect of the present invention includes collecting the complex containing the separated target cells from the outlet, or collecting the separated target cells from the outlet. When a complex of the target cell and the target capture substance is generated, the complex is collected from the outlet, and when a complex of the non-target cell and the target capture substance is generated, the target cell is discharged from the outlet. to recover. These can be recovered by using a known method as appropriate, such as attaching a tube to the discharge port and sucking with a pump.

2-4. Step of Removing Microaggregates In the method for separating cells according to one aspect of the present invention, when separating target cells from a blood sample containing microaggregates, the microaggregates described later are removed before the separation step. It is preferable to add that. Removal of microaggregates can be performed as a pretreatment of blood samples, but when combined with the above method for cell separation, after complex formation and the basic structure of the DLD microchannel of the cell separation device. By adding the removal of these microaggregates before introducing the blood sample into the blood sample, the separation of target cells can be effectively performed.

That is, the cell separation device may have a microaggregate removing device between the sample inlet and the basic structure of the DLD microchannel.

3. 3. Principle of removing microaggregates in cell separation method 3-1. Micro-aggregate removing device FIG. 8 is a schematic diagram illustrating a basic structure 110 of a micro-aggregate removing device used for removing micro-aggregates in one aspect of the present invention. The structure 111 is installed in a straight line in the vertical direction with respect to the flow direction of the fluid in the direction of the arrow, and the sample, that is, the microaggregate 112 in the blood sample is captured by the structure 111 by the flow. By providing a plurality of these structures 111 in the horizontal direction with respect to the flow direction at regular intervals, the microaggregates are sequentially captured by the structure, and the microaggregates in the blood sample are removed as they go downstream in the flow direction. Will be done.

FIG. 9 is an explanatory diagram showing that the microaggregate removing device is composed of pillar structures 121 at regular intervals. By arranging the pillar structure 121 according to a certain rule, a flow bypassing the portion where the microaggregates are sequentially captured is generated, and the microaggregates can be efficiently removed sequentially. By diverting the flow in this way, the loss of the target cells and the like can be avoided, and the treatment can be performed without a decrease in the flow velocity.

The shape of this pillar may be, for example, a cylindrical structure, but the shape is not particularly limited as long as it is a structure capable of capturing the target microaggregates. For example, it is possible to have a polygonal prism structure in which the horizontal cross-sectional shape is a rhombus. The diameter of each pillar can be, for example, about 5 to 30 μm.

Further, in this pillar structure, the basic structure of the shape may be continuously arranged at regular intervals, and the shape and arrangement can be gradually changed and installed according to the purpose. As described above, the pillar structures are not limited to being arranged at regular intervals throughout the microaggregate removing device, and may be installed randomly. For example, the installation interval may be wide in the upstream portion in the flow direction, and the installation interval may be gradually narrowed toward the downstream portion. Further, the pillar structure may be installed only in a part of the micro-aggregate removing device, or a pillar structure having a different basic structure may be installed in a specific part of the micro-aggregate removing device. Further, the width of the entire flow path does not have to be uniform, and the flow path width may be narrowed in the middle if necessary.

If the installation interval of these pillars is too narrow, microaggregates will be sequentially captured from the upstream part in the flow direction, so clogging is likely to occur preferentially from the upstream part. For this reason, when large micro-aggregates are present in the sample or a large amount of micro-aggregates are contained, it is preferable to widen the pillar installation interval in the upstream portion in the flow direction, and the downstream in the flow direction. It is preferable that the installation interval is gradually narrowed as compared with the upstream portion so as to capture the small agglomerates that could not be captured in the upstream portion in the flow direction. If the pillar installation interval in the upstream part is narrow from the beginning, the captured microaggregates may clog the microaggregate removing device, making processing impossible, or trapping target cells, etc. This can result in loss.

If the pillar structure is arranged linearly with respect to the flow direction, there is a possibility that microaggregates will reach the downstream as they are without being trapped. Therefore, for the purpose of capturing and removing the microaggregates of the present embodiment, it is preferable that the pillar structure is not arranged linearly with respect to the flow direction. For example, the pillars can be arranged so as to be offset by one row each time 5 to 30 pillars are installed in the flow direction with respect to the flow direction, and 10 to 20 pillars are installed in the flow direction. It is preferable that the arrangement is shifted by one row for each row in order to maintain the flow and efficiently separate the microaggregates.

The pillar installation interval is preferably larger than or equal to the size of the target cells (including the complex of the target cells and the target capture substance) contained in the blood sample. If the installation interval is smaller than the size of the target cell, the target cell (complex of the target cell and the target capture substance) is captured between the pillars, resulting in loss of the target cell.

Generally, the size of cells contained in blood is about 30 μm at the largest, so if the purpose is to separate all cells in blood, the size should be larger than this. For example, when the purpose is to separate red blood cells or platelets and it is necessary to remove the white blood cells, the size can be set to 8 μm or more, which is the size for dividing these cells.

It is also possible to remove the microaggregates after binding the carrier substance to the target cells in the blood sample in advance to form a complex. In this case, since the complex of the target cell and the carrier substance is trapped between the pillars and the target cell is lost, it is preferable that the pillar installation interval is equal to or larger than the size of this complex. Specifically, examples of the carrier substance include beads and the like capable of labeling an antibody and the like, but the carrier substance is not limited to this, and any substance may be considered as the carrier substance.

For example, when a carrier substance having a size of 30 μm is used to form a complex with cells having a size of about 20 μm, the pillar installation interval is preferably 50 μm or more.

Further, it is preferable that the pillar installation interval has a portion of 200 μm or less. Some of the microaggregates are relatively small in size. Therefore, if the pillar installation intervals of the micro-aggregate removing device are all set to 200 μm or more, the micro-aggregates may not be completely removed. However, it is not necessary that all the pillar installation intervals are 200 μm or less, and a pillar structure having a larger installation interval can be provided depending on the purpose.

As described above, in the micro-aggregate removing device, the micro-channel structure serving as the micro-aggregate removing mechanism (sometimes referred to as “second micro-channel structure” in the present specification) has an interval larger than 30 μm. It is preferable that the pillars are installed in the above, and it is preferable that the pillars are installed at intervals of 200 μm or less. The pillar installation interval of this microchannel structure depends on the target cell (complex of the target cell and the carrier substance) and the size of the microaggregate to be removed, but when removing the microaggregate in blood. The pillar installation interval may be set, for example, 50 μm to 200 μm, preferably 70 μm to 170 μm, and more preferably 90 μm to 150 μm. The pillar installation interval means an average interval when two or more pillars are installed at different intervals in the microchannel structure.

On the other hand, the complex in which the target cell and the carrier substance are bound is larger by the size of the carrier substance as compared with the target cell. Therefore, when this complex is formed in blood, in order to remove microaggregates in blood and recover this complex (prevent clogging), the pillar installation interval is a carrier. It is preferable to increase the size of the substance. For example, when a carrier substance having a diameter of 30 to 50 μm is used, the pillar installation interval may be set to, for example, 80 μm to 250 μm, preferably 100 μm to 230 μm, and more preferably 120 μm to 220 μm. preferable.

The microaggregate removing device may be provided with a plurality of second microchannel structure portions as described above. By providing a plurality of second microchannel structural parts, the efficiency of removing microaggregates is increased, but the efficiency of recovery of target cells (complex of the target cells and carrier substance) is improved, and the structure of the device is simplified. From the viewpoint of conversion, it is preferable to provide two microchannel structure portions.

FIG. 10 is a schematic diagram illustrating a microagglutination removing device according to an embodiment of the present invention. When the microaggregate removing device 130 includes two second microchannel structural portions, it is preferable to provide a portion 46 for converging the flow width in the middle (FIG. 10). This portion 46 is narrower than the second microchannel structure portion and has no pillars installed. In this portion 46, microaggregates that have passed through the first second microchannel structural portion (structural portion α) are accumulated and flow to the next second microchannel structural portion (structural portion β). Go (Fig. 10, Fig. 11A). By providing the portion 46 for converging such a flow width, microaggregates can be efficiently removed.

As described above, when the micro-aggregate removing device 130 includes two or more second microchannel structural portions, in the micro-aggregate removing device 130, the second micro on the downstream side where the target cells are discharged. As described above, the pillar installation interval of the flow path structural portion (structural portion β) may be set to 80 μm to 250 μm, preferably 100 μm to 230 μm, and more preferably 120 μm to 220 μm. Then, the pillar installation interval of the second microchannel structural portion (structural portion α) on the upstream side into which the blood sample containing the target cell is introduced is set to that of the microchannel structural portion (structural portion β) on the downstream side. By setting the same level or making it wider than that, it is possible to process a relatively large number of blood samples while increasing the efficiency of removing microaggregates and preventing clogging due to microaggregates.

4. Removal of microaggregates Removal of microaggregates is performed by a method of removing microaggregates from a blood sample in a continuous flow of liquid as described above.

Microaggregates in blood are derived from agglutinated fibrin, other denatured proteins, fat and the like. Since the microaggregates are particularly viscous, they are easily trapped by the pillar structure of the microaggregate removing device, and once trapped by the pillar structure, they are less likely to be detached and flow out downstream in the flow direction. ..

In the cell separation method according to the embodiment of the present invention, a blood sample or a mixed solution of a blood sample and a buffer solution is flowed through a microaggregate removing device including a second microchannel structure, and the second microchannel structure is used. It may include removing microaggregates from the blood sample in a continuous stream of liquid by passing through the structure and capturing the microaggregates contained in the blood sample.

When a micro-aggregate removing device is used in the cell separation method according to the embodiment of the present invention, a blood sample and a buffer solution are added to the micro-aggregate removing device. At this time, the buffer solution may be poured in advance, and then the blood sample may be added, or the blood sample may be mixed with the buffer solution, diluted, and then added. Then, as described above, by capturing the microaggregates, the microaggregates in the blood sample are removed, and the blood sample containing the target cells is collected from the outlet of the microaggregate removing device. If a liquid feeding tube or the like is provided at the discharge port of the micro-aggregate removing device, the liquid is collected through the tube or the like. Further, as described above, when the system is connected to the cell separation device to form a continuous system, that is, when the cell separation device contains a microaggregate removing device, the blood sample collected by removing the microaggregates After that, cell separation is performed. The amount of blood sample relative to the buffer solution is not particularly limited. Further, the speed at which the cell suspension flows is not particularly limited. Regarding this flow velocity, the speed can be appropriately adjusted by installing a pump or the like at either one or both of the introduction port and the discharge port of the cell separation device.

The method for separating cells according to the embodiment of the present invention may include a method used for pretreatment when accurately collecting the target cells contained in the blood sample, without loss of the target cells in the blood sample. Moreover, by accurately removing the microaggregates in the blood sample, the target cells can be recovered more accurately in the subsequent cell separation.

When the method for separating cells according to the embodiment of the present invention includes removing microaggregates from a blood sample, it may include an embodiment in which an anticoagulant reagent is added. In addition to the presence of microaggregates already in the blood sample, there is a possibility that new microaggregates may be generated in the process of processing blood. Therefore, although the existing microaggregates can be removed by the method according to the above aspect of the present invention, they are included in the blood sample in order to prevent new microaggregates from being generated during the treatment of blood. Anti-blood coagulation reagents may be added to the composition of the dilution buffer. The blood from which the microaggregates have been removed by adding an anticoagulant reagent to remove the microaggregates is used for a subsequent blood test or various treatments (including separation of blood components, etc.). Can be done.

Examples of anticoagulant reagents include sodium citrate, EDTA, heparin, thrombin inhibitors such as PPACK, and the like. Among these, it is preferable to use a thrombin inhibitor, and it is more preferable to use a reagent such as PPACK.

Sodium citrate, EDTA, etc. inhibit calcium ions and indirectly inhibit the blood coagulation action, but it is more effective to directly inhibit thrombin, which is a coagulation factor. As the thrombin inhibitors, heparin, inhibits only free _{thrombin, PPACK (C 21 H 31 ClN} 6 O 3 .2HCl) [1- (2-Amino-3-phenylpropanoyl) -N- [1-chloro -6- (diaminomethylideneamino) -2-oxohexan-3-yl] pyrrolidine-2-carboxamide] is more effective because it acts on both free and bound thrombin.

The removal of microaggregates is the removal of microaggregates that already exist in blood, and by using a thrombin inhibitor such as PPACK in combination, the formation of new microaggregates in blood is suppressed and the microaggregates are removed into the blood. It is possible to remove the microaggregates that already exist.

In the cell separation method according to the embodiment of the present invention, the microaggregate removing device having a second microchannel structure and a diluted buffer solution containing thrombin inbiter are used in combination. And in the DLD microchannel, clogging of microaggregates can be suitably cleared. As a result, the removal method using the micro-aggregate removing device enables efficient and continuous removal of micro-aggregates and separation of cell components.

Although the embodiments of the present invention have been described in detail above, the present invention is not limited to the above embodiments and is included in the present invention even if there are changes to the extent that the gist of the present invention is not deviated.

The present invention includes the following aspects as another aspect.
[12] The blood sample containing the target cells is placed in an environment of 10 ° C. or higher and 40 ° C. or lower for a period of 1 hour or more and 3 days or less.
Deterministic transverse substitution method The blood sample is added from the sample introduction port to a cell separation device provided with a separation area having a microchannel structure, a sample inlet and an outlet, and the blood sample is introduced into the cell separation device. That and
Separation of the target cells from the blood sample in a continuous stream of liquid containing the blood sample.
Collecting the separated target cells from the outlet and
A method for separating cells including.
[13] The method for separating cells according to [12], wherein the period is 3 hours or more and 2 days or less.
[14] The method for separating cells according to [12], wherein the period is 6 hours or more and 1 day or less.
[15] The method for separating cells according to any one of [12] to [14], wherein the temperature of the blood sample is 15 ° C. or higher and 40 ° C. or lower.
[16] The method for separating cells according to any one of [12] to [14], wherein the temperature of the blood sample is 20 ° C. or higher and 40 ° C. or lower.
[17] The method for separating cells according to any one of [12] to [14], wherein the temperature of the blood sample is 25 ° C. or higher and 37 ° C. or lower.
[18] The method for separating cells according to any one of [12] to [17], further comprising adding and flowing a buffer solution from the buffer inlet of the cell separation device.
[19] The method for separating cells according to [18], wherein the temperature of the buffer solution is 10 ° C. or higher and 40 ° C. or lower.
[20] The method for separating cells according to any one of [12] to [19], further comprising removing microaggregates contained in the blood sample before the introduction of the blood sample.
[21] The blood sample further contains non-target cells.
Prior to introduction into the cell separation device, a target capture substance that recognizes a characteristic structure existing on the surface of the target cell is added to the blood sample, and a complex of the target cell and the target capture substance is added. Including generating
Separation of the target cells is to separate the complex having a size equal to or greater than a determined threshold, and cells having a size smaller than the threshold move with the flow of the blood sample and The complex having a size equal to or larger than the threshold value is separated by being displaced and moved in an oblique direction with respect to the flow.
The method for separating cells according to any one of [12] to [20], wherein the target cells are collected as the complex in the collection.
[22] The blood sample contains target cells and non-target cells.
Prior to introduction into the cell separation device, a target capture substance that recognizes a characteristic structure existing on the surface of the target cell is added to the blood sample, and a composite of the target cell and the target capture substance is added. Including producing the body
Separation of the target cells is a step of separating the complex having a size equal to or larger than the determined threshold value, and the cells having a size smaller than the threshold value move together with the flow of the blood sample and The complex having a size equal to or larger than the threshold value is separated by being displaced and moved in an oblique direction with respect to the flow.
The method for separating cells according to any one of [12] to [20], which collects the target cells.
[23] The target capture substance is characterized by comprising a conjugate of a target capture molecule having a characteristic structure existing on the surface of the target cell or the non-target cell and a substance carrying the target capture molecule. The method for separating cells according to claim [21] or [22].
[24] The method for separating cells according to [23], wherein the target capture molecule is an antibody, a peptide aptamer, a lectin, an intercellular adhesion molecule, a sugar chain, or a cell-recognizable polymer.
[25] The method for separating cells according to [23] or [24], wherein the substance supporting the target capture molecule is polystyrene or latex.

Hereinafter, this embodiment will be described in detail with reference to examples. However, in reality, the present embodiment is not limited to the following examples.

(Example 1)
(1) Manufacture of an integrated separation device A microaggregate removing device in which two second microchannel structural parts (structural part α: pillar spacing of 200 μm, structural part β: pillar spacing of 150 μm) are connected in series. 130 was designed (Fig. 10). The outer side surfaces of the structural portion α and the structural portion β are not provided with pillars within 80 μm from the wall surface of the flow path in order to prevent clogging at the end of the flow path. Further, a portion for converging the flow width is provided between the structural portion α and the structural portion β. An integrated cell separation device was produced as a design in which the microaggregate removing device 130 having these two structural parts was integrated with the separation device having the basic structure 20 of the DLD microchannel having a separation threshold of 30 μm ( 11A and 11B).

(2) Preparation of blood sample After blood collection, whole blood is stored at room temperature (25 ° C.) for 4 hours, and then diluted 2-fold with 1% BSA at room temperature (25 ° C.) and PBS buffer containing 5 mM EDTA. A sample solution was prepared by adding a thrombin inhibitor (PPACK, manufactured by abcam) so that the final concentration was 80 μM. In the sample solution, polystyrene beads (CD326 S-puliBade® anti-hu, manufactured by purriSelect Life Science) having a diameter of 32 μm as a target capture substance and carrying an anti-human CD326 antibody were added per 1 mL of whole blood. It was added so as to be 1 × 10 ⁵ pieces.

The integrated cell separation device was previously filled with a PBS buffer containing 1% BSA and 5 mM EDTA at room temperature (25 ° C.). Then, the sample solution was sent to the sample introduction port 31 at a rate of 50 μl / min using a syringe pump. At the same time, a PBS buffer containing 1% BSA and 5 mM EDTA was sent to the buffer inlet 32 at a rate of 500 μl / min, and recovery was performed from the respective recovery ports (reference numerals 33 and 34). The entire process from the introduction of the blood sample to the collection was carried out in a room whose temperature was adjusted to room temperature (25 ° C.).

The blood sample prepared in (2) is introduced into the integrated cell separation device, and the flow of the blood sample on the structural part α, the structural part β, the introduction port side, the intermediate part, and the discharge port side of the cell separation mechanism is changed. Observation was performed with a transmission type optical microscope.

As a result, it was confirmed that cells having a size of less than 30 μm in the blood sample were collected from the second outlet (reference numeral 34) without causing clogging in the device (FIG. 13 [the day]). In addition, the target trapped substance was recovered from the first outlet (reference numeral 33).

(Example 2)
The structure part α, the structure part β, and the cells are the same as in Example 1 except that the blood sample is introduced into the integrated cell separation device after collecting the blood and storing the whole blood at room temperature (25 ° C.) for 24 hours. The blood flow on the introduction port side (top), middle part (middle), and discharge port side (tail) of the separation mechanism was observed with a transmission optical microscope.
As a result, it was confirmed that cells having a size of less than 30 μm in the blood sample were collected from the second outlet (reference numeral 34) without causing clogging in the device (FIG. 13 [1 day later]). .. In addition, the target trapped substance was recovered from the first outlet (reference numeral 33).

(Comparative Example 1)
After collecting blood, the whole blood is stored at 4 ° C. for 4 hours, and then an integrated cell separation device is also introduced. In the same manner as in Example 1, the structural part α, the structural part β, and the cell separation mechanism The blood flow on the inlet side, the middle portion, and the outlet side was observed with a transmission type optical microscope.
As a result, it was confirmed that the blood aggregated immediately after the introduction of the blood sample (Fig. 14A). In addition, it was confirmed that blood agglutination further increased 40 minutes after the introduction of the blood sample (FIG. 14B).

The cell separation method of the present invention can be used for research use, diagnostic use, and cell separation and purification in the manufacture of pharmaceutical products and the like.

1 Cell suspension 2 Target cell 3 Non-target cell 4 Mixed solution 5 Complex 10 Target capture molecule 11 Substance that carries the target capture molecule 12 Target capture substance 20 Basic structure of DLD microchannel 21 Obstacle structure 22 Constant size Particles larger than or equal to 23 Particles smaller than a certain size 30 Cell separation device with DLD microchannel 31 Sample inlet 32 Buffer inlet 33 First outlet 34 Second outlet 35 Channel space 36 Channel Structure part 37 Plane structure part 38 Partition 39 Branch part 40 Cell separation device with liquid feeding part and collecting part 41 Sample liquid feeding part 42 Buffer liquid feeding part 43 First collecting part 44 Second collecting part 110 Basic structure 111 Structure 112 Micro-aggregate 121 Pillar structure 131 Inlet 132 Outlet

Claims (11)

1. The blood sample containing the target cells is not placed in an environment of 4 ° C. or lower, and the blood sample is added from the sample introduction port to a cell separation device having a separation area having a microchannel, a sample introduction port and a discharge port, and the blood sample is poured. Introducing the blood sample into the cell separation device and
   Separation of the target cells from the blood sample in a continuous stream of liquid containing the blood sample.
   Collecting the separated target cells from the outlet and
   A method for separating cells including.
2. The cell separation method according to claim 1, wherein not placing in an environment of 4 ° C or lower means placing in an environment of 10 ° C or higher and 40 ° C or lower.
3. The cell separation method according to claim 1 or 2, further comprising adding and flowing a buffer solution from the buffer inlet of the cell separation device.
4. The method for separating cells according to claim 3, wherein the temperature of the buffer solution is 10 ° C. or higher and 40 ° C. or lower.
5. The method for separating cells according to any one of claims 1 to 4, wherein the blood sample is blood diluted with a diluent.
6. The method for separating cells according to any one of claims 1 to 5, wherein the separation area having the microchannel is a separation area having a deterministic transverse substitution method microchannel structure.
7. The blood sample further contains unintended cells
   Prior to introduction into the cell separation device, a target capture substance that recognizes a characteristic structure existing on the surface of the target cell is added to the blood sample, and a complex of the target cell and the target capture substance is added. Including generating
   Separation of the target cells allows cells having a size smaller than the threshold to move with the flow of the blood sample, and the complex having a size greater than or equal to the threshold to move obliquely with respect to the flow. Is to separate by doing
   The method for separating cells according to any one of claims 1 to 6, wherein the target cells are recovered as the complex in the recovery.
8. The blood sample further contains unintended cells
   Prior to introduction into the cell separation device, a target capture substance that recognizes a characteristic structure existing on the surface of the target cell is added to the blood sample, and a composite of the target cell and the target capture substance is added. Including producing the body
   The cells having a size smaller than the threshold for separating the target cells move with the flow of the blood sample, and the complex having a size equal to or larger than the threshold is displaced obliquely with respect to the flow. The method for separating cells according to any one of claims 1 to 6, wherein the cells are separated by moving.
9. A claim characterized in that the target capture substance comprises a conjugate of a target capture molecule having a characteristic structure existing on the surface of the target cell or a non-target cell and a substance carrying the target capture molecule. Item 7. The method for separating cells according to Item 7.
10. The method for separating cells according to claim 9, wherein the target capture molecule is an antibody, a peptide aptamer, a lectin, an intercellular adhesion molecule, a sugar chain, or a cell-recognizable polymer.
11. The method for separating cells according to claim 9 or 10, wherein the substance supporting the target capture molecule is polystyrene or latex.

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