JP2013142540A - Cell segregation device and cell segregation method using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a cell segregation device in which blood platelets can be segregated accurately from among a plurality of kinds of cells contained in a blood-derived sample in simple configuration and in the case where circulating tumor cells (CTC) are contained in the blood-derived sample, the CTC are suppressed from being removed erroneously, and a cell segregation method using the same.SOLUTION: A cell segregation device is provided for segregating desired cells from among a plurality of kinds of cells contained in a blood-derived sample. The cell segregation device comprises a main body part and in the main body part, at least a blood platelet trap region is provided for trapping blood platelets in the blood-derived sample. In the blood platelet trap region, a substance which interacts with the blood platelets is immobilized to the main body part.

Description

  The present invention relates to a cell separation device for separating desired cells from a plurality of types of cells contained in a blood-derived sample, and a cell separation method using the same.

  2. Description of the Related Art Conventionally, cytodiagnosis has been performed in which a specific cell is taken out from a blood-derived sample and observed to perform the presence / absence of a lesion, pathological diagnosis of a lesion, clinical diagnosis, and the like.

  In particular, as one of the methods for observing the presence or absence of blood circulating cancer cells (hereinafter also referred to as CTC) in a blood-derived sample or the state of CTC, a plurality of types of cells in a blood-derived sample are separated by specific gravity, A separation layer that can contain CTCs is taken out and developed into a planar shape, and the cells are fluorescently stained and then observed with an optical detection means.

  A method for specific gravity separation of cells, particularly CTC, in a blood-derived sample is as follows. For example, as disclosed in Patent Document 1, first, a separation medium smaller than the specific gravity of platelets and some white blood cells is placed in a test tube. A blood-derived sample is added to this, and this is set in a centrifuge and a centrifugal force is applied to separate a plurality of types of cells contained in the blood-derived sample by the difference in specific gravity.

  The specific gravity of major components contained in the blood-derived sample is such that plasma <platelet <white blood cell <red blood cell, and red blood cell has the largest specific gravity. Therefore, as shown in FIG. In the test tube 100, the red blood cell 208 is located in the lowest layer. Since the specific gravity of blood circulating cancer cells (CTC) 300 is approximately the same as that of platelets 204 and leukocytes 206 or even smaller than that of platelets 204 and leukocytes 206, the blood-derived sample 200 contains CTC300. The CTC 300 is considered to be located in the same layer as the platelets 204 and white blood cells 206 or in the layer of the plasma 202 above the platelets 204 and white blood cells 206.

  For this reason, if all layers above this layer, including some red blood cells 208 after centrifugation, are sampled and detected, it should be possible to detect CTC 300 without leaking.

JP 2002-536635 Gazette

  By the way, the sampled sample contains platelets 204 together with the CTC 300. Since the platelet 204 is much larger than the CTC 300, the CTC 300 may be buried in the platelet 204 when the sampled sample is developed in a planar shape, which may cause a detection loss of the CTC 300. It was. For this reason, Patent Document 1 discloses a method of washing and removing platelets by multiple times of centrifugation, but there is a problem that cells containing CTC are lost by the centrifugation operation.

  Note that the development area of the sampled sample may be increased so that the CTC 300 is not buried in the platelets 204. However, in this case, since the range to be detected is expanded this time, it takes a lot of time to detect the CTC 300. In addition, it is necessary to increase the size of the apparatus, which is not realistic.

  Furthermore, since CTC300 may exist in the form of a complex with platelet 204, if platelet 204 is simply separated, this complex is also separated together, resulting in a complex CTC. May not be detected.

  The present invention has been made in view of such a current situation, and it is possible to accurately separate platelets from a plurality of types of cells contained in a blood-derived sample with a simple configuration, and to obtain a blood-derived sample. Provided with a cell separation device that suppresses erroneous removal of blood circulation cancer cells (CTC) when blood circulation cancer cells (CTC) are contained therein, and a cell separation method using the same The purpose is to do.

The present invention was invented to solve the problems in the prior art as described above,
The cell separation device of the present invention comprises:
A cell separation device for separating desired cells from a plurality of types of cells contained in a blood-derived sample,
The cell separation device includes a main body,
The main body is provided with a platelet trap region for trapping at least platelets in the blood-derived sample,
The platelet trap region is characterized in that a substance that interacts with the platelets is immobilized on the main body.

  In this way, if a platelet trap region in which a substance that interacts with platelets is immobilized on the main body is provided, many platelets can be removed from a blood-derived sample with a simple configuration.

  Since platelets and platelet / CTC complexes have different affinity, the platelet / CTC complex is hardly trapped in this platelet trap region.

  Therefore, if the remaining blood-derived sample from which most of the platelets have been separated is developed in a planar shape, it is unlikely that CTC will be buried in the platelets that have been produced in the past, and the complex of platelets and CTCs is also detected. Therefore, CTC can be detected with high accuracy.

Moreover, the cell separation device of the present invention comprises:
The main body is plate-shaped,
The platelet trap region is provided on a part of the upper surface of the plate-shaped main body.

  Thus, if the main body is plate-shaped, the blood-derived sample can be easily supplied to the platelet trap region, so that the blood-derived sample can be supplied to the platelet trap region with a simple operation.

Moreover, the cell separation device of the present invention comprises:
The platelet trap region is provided to be biased to one side of the upper surface of the main body.

  With this configuration, when a blood-derived sample is supplied to the platelet trap region, the platelet trap region can be positioned upstream of the flow, and the blood-derived sample is reliably supplied to the platelet trap region. can do.

Moreover, the cell separation device of the present invention comprises:
The platelet trap region provided on a part of the upper surface of the main body portion,
It is characterized by a flat planar shape.

  With this configuration, when a blood-derived sample is supplied to the platelet trap region, the blood-derived sample can be easily spread throughout the platelet trap region, and platelets are efficiently trapped in the platelet trap region. be able to.

Moreover, the cell separation device of the present invention comprises:
A blood circulation cancer cell (CTC) trap region for trapping blood circulation cancer cells (CTC) and other cells is further provided on the upper surface of the main body.

  If a blood circulation cancer cell (CTC) trap region is provided in this way, a single cell separation device can trap platelets and CTCs, so that CTC detection loss can be suppressed as much as possible. CTC can be detected.

Moreover, the cell separation device of the present invention comprises:
In the blood circulation cancer cell (CTC) trap region,
A plurality of grooves are juxtaposed in a line from one side of the main body to the other side.

  If the groove is provided in this manner, CTC and other cells can be highly integrated and aligned in the groove, which can contribute to miniaturization of the device. Further, when detecting CTC, it is only necessary to detect the CTC by moving the detection means along the groove forming direction, so that the time required for detection can be shortened and the detection accuracy can be increased.

Moreover, the cell separation device of the present invention comprises:
The groove width is in the range of 1 to 100 μm and the groove depth is in the range of 1 to 100 μm.

  Within such a range, the remaining cells including CTC can be surely aligned and expanded in the groove.

Moreover, the cell separation device of the present invention comprises:
The groove has a V-shaped cross section.

  With such a shape, the remaining cells including CTC can be surely aligned and expanded in the groove while ensuring the fluidity of the blood-derived sample.

Moreover, the cell separation device of the present invention comprises:
The main body is a bottomed tubular,
The platelet trap region is provided circumferentially on a part of the inner peripheral surface of the bottomed tubular main body.

  In this way, if the main body is a bottomed tubular body, for example, by simply rotating the bottomed tubular main body containing a blood-derived sample and stirring the blood-derived sample, Many of the platelets can be trapped.

  Therefore, platelets in the blood-derived sample can be efficiently trapped in the platelet trap region by a simple operation.

Moreover, the cell separation device of the present invention comprises:
The main body has a well shape having a plurality of recesses,
The platelet trap region is provided on the upper surface of the well-shaped main body portion excluding the concave portion.

  In this way, if the main body is in a well shape, the platelet in the blood-derived sample is placed in the platelet trap region simply by shaking the well-shaped main body containing the blood-derived sample and stirring the blood-derived sample. Can be trapped. At the same time, a blood-derived sample from which platelets have been removed can be dropped into the recess and collected.

  Therefore, platelets in a blood-derived sample can be efficiently trapped in a platelet trap region by a simple operation, and the blood-derived sample excluding platelets can be collected at the same time.

Moreover, the cell separation device of the present invention comprises:
The diameter of the recess is in the range of 1 to 500 μm.

  Within such a range, the blood-derived sample excluding platelets can be reliably dropped and collected in the recess.

Moreover, the cell separation device of the present invention comprises:
The blood-derived sample is a sample after red blood cell separation processing has been performed in advance.

  Thus, if the red blood cells which occupy most of the blood cells are previously separated, it is possible to further easily separate the platelets from the blood-derived sample.

Moreover, the cell separation device of the present invention comprises:
The red blood cell separation process is performed by density gradient centrifugation.

  By using the density gradient centrifugation method in this way, blood cells contained in a blood-derived sample can be surely arranged in layers in order of specific gravity. Therefore, the red blood cell separation process can be performed with high accuracy.

Moreover, the cell separation device of the present invention comprises:
The specific gravity of the separation medium used in the density gradient centrifugation method is
It is smaller than red blood cells and larger than white blood cells.

  If the separation medium has a specific gravity adjusted as described above, when the density gradient centrifugation is performed, the blood-derived sample is at least two layers: a layer containing many red blood cells and a layer containing many cells other than red blood cells. Since the layers can be separated, most of the red blood cells can be removed from the blood-derived sample by taking out only the layer containing many cells other than red blood cells.

Moreover, the cell separation device of the present invention comprises:
The substance that interacts with the platelets is a substance that interacts with a membrane glycoprotein expressed on the cell membrane of the platelets.

  With such a substance, platelets can be effectively trapped in the platelet trap region.

Moreover, the cell separation device of the present invention comprises:
The substance that interacts with the membrane glycoprotein is an antibody that interacts with the membrane glycoprotein.

  With such an antibody, platelets can be effectively trapped in the platelet trap region.

Moreover, the cell separation device of the present invention comprises:
The substance that interacts with the membrane glycoprotein is a substance having a dissociation constant (K _D ) in the range of 10 ⁻⁷ to 10 ⁻⁶ M.

  In this way, if the substance has a low affinity (affinity), even if the platelet-CTC complex is trapped in the substance that interacts with the platelet in the platelet trap region, it is derived from the blood to be supplied thereafter. The trap can be released only by the force of the sample flow.

Moreover, the cell separation method of the present invention comprises:
A cell separation method for separating desired cells from a plurality of types of cells contained in a blood-derived sample,
The cell separation method comprises:
A cell separation device having a platelet trap region in which a substance that interacts with platelets in the blood-derived sample is immobilized is prepared on the main body, and the blood-derived device is placed on the platelet trap region of the cell separation device A step of trapping the platelets in the blood-derived sample in the platelet trap region;
It is characterized by having at least.

  With such a method, most of the platelets in the blood-derived sample can be reliably trapped in the platelet trap region.

Moreover, the cell separation method of the present invention comprises:
In the step of trapping the platelets,
When supplying the blood-derived sample on the platelet trap region, the flow rate of the blood-derived sample is adjusted using a flow rate adjusting means so that the blood-derived sample remains on the platelet trap region. And

  By adjusting the flow rate of the blood-derived sample using the flow rate adjusting means in this way, the interaction with platelets can be sufficiently exerted in the platelet trap region, and many platelets can be efficiently trapped in the platelet trap region. Can do.

Moreover, the cell separation method of the present invention comprises:
In the cell separation device prepared in the step of trapping the platelets,
A blood circulation cancer cell (CTC) trap region for trapping blood circulation cancer cells (CTC) and other cells is further provided on the upper surface of the main body.
Following the step of trapping the platelets,
Supplying the blood-derived sample to the blood circulation cancer cell (CTC) trap region, and trapping the blood circulation cancer cell (CTC) and other cells in the blood circulation cancer cell (CTC) trap region When,
It is characterized by having.

  With such a method, platelet trapping and CTC trapping can be performed using a single cell separation device, so that detection loss of CTC can be suppressed as much as possible, and CTC can be detected with high accuracy.

Moreover, the cell separation method of the present invention comprises:
A flow rate of the blood-derived sample is in a range of 100 to 1000 μl / sec.

  With such a relatively high flow rate, even if a platelet-CTC complex is trapped by a substance that interacts with platelets in the platelet trap region, the trap is performed with the strength of the blood-derived sample flow. Can be released. Even at a low flow rate, the complex of platelets and CTC can be released by adding a fluid for a long time.

Moreover, the cell separation method of the present invention comprises:
Following the step of trapping the platelets,
Developing the remaining blood-derived sample in which the platelets are trapped in a planar shape;
It is characterized by having.

  With such a method, the remaining cells including the CTC can be developed in a planar shape without the CTC being buried in the platelets, and the CTC can be detected with high accuracy.

Moreover, the cell separation method of the present invention comprises:
The blood-derived sample is a sample after red blood cell separation processing has been performed in advance.

  Thus, if the red blood cells which occupy most of the blood cells are previously separated, it is possible to remove more of the platelets in the blood-derived sample more reliably.

Moreover, the cell separation method of the present invention comprises:
The red blood cell separation process is performed by density gradient centrifugation.

  By using the density gradient centrifugation method in this way, blood cells contained in a blood-derived sample can be surely arranged in layers in order of specific gravity. Therefore, the red blood cell separation process can be performed with high accuracy.

Moreover, the cell separation method of the present invention comprises:
The specific gravity of the separation medium used in the density gradient centrifugation method is
It is smaller than red blood cells and larger than white blood cells.

  If the separation medium has a specific gravity adjusted as described above, when the density gradient centrifugation is performed, the blood-derived sample is at least two layers: a layer containing many red blood cells and a layer containing many cells other than red blood cells. Since the layers can be separated, if only the layer containing many cells other than red blood cells is taken out, only red blood cells can be almost certainly removed from the blood-derived sample.

Moreover, the cell separation method of the present invention comprises:
The substance that interacts with the platelets is a substance that interacts with a membrane glycoprotein expressed on the cell membrane of the platelets.

  Such a substance can surely trap most of the platelets in the platelet trap region.

Moreover, the cell separation method of the present invention comprises:
The substance that interacts with the membrane glycoprotein is an antibody that interacts with the membrane glycoprotein.

  Such an antibody can surely trap most of the platelets in the platelet trap region.

Moreover, the cell separation method of the present invention comprises:
The substance that interacts with the membrane glycoprotein is a substance having a dissociation constant (K _D ) in the range of 10 ⁻⁷ to 10 ⁻⁶ M.

  If a substance with such a low affinity (affinity) is used, even if a complex of platelets and blood circulating cancer cells (CTC) is trapped in a substance that interacts with platelets in the platelet trap region, Thereafter, the trap can be released with the strength of the flow when supplying the blood-derived sample.

  According to the present invention, a platelet trap region in which a substance that interacts with platelets is immobilized is provided in the main body, and platelets are selected from a plurality of types of cells contained in a blood-derived sample. Can be separated with high accuracy, and when blood-circulating cancer cells (CTC) are contained in a blood-derived sample, it is possible to prevent the blood-circulating cancer cells (CTC) from being erroneously removed. Cell separation device and cell separation method using the same can be provided.

FIG. 1 is a perspective view showing a first embodiment of the cell separation device of the present invention. FIG. 2 is a cross-sectional view taken along line AA of the cell separation device of the present invention shown in FIG. FIG. 3 is a partially enlarged view for explaining the dimensions of the groove of the cell separation device of the present invention. FIG. 4 is an enlarged cross-sectional view of the groove of the cell separation device of the present invention. FIG. 4 (a) shows only one side of the groove inclined, and FIG. 4 (b) shows a rounded end of the groove. FIG. 4C shows a groove formed in a U-shaped cross section. FIG. 5 is a schematic view showing a second embodiment of the cell separation device of the present invention. FIG. 6 is a schematic view showing a third embodiment of the cell separation device of the present invention. FIG. 7 is a view showing a separated state of blood components when blood is centrifuged.

  Hereinafter, embodiments of the present invention will be described in more detail based on the drawings.

  The cell separation device according to the first to third embodiments of the present invention is used for separating platelets from a plurality of types of cells contained in a blood-derived sample.

  The cell separation method of the present invention is a method for separating platelets from a plurality of types of cells contained in a blood-derived sample.

  In the present specification, “blood-derived sample” means blood mixed with a blood collection additive such as a blood coagulation inhibitor, diluted blood, a specific blood other than intact blood collected from a living body. Blood from which components have been removed in advance, blood containing CTC, and blood in which CTC has been fluorescently stained in advance are also included.

  Here, for example, when preparing a blood-derived sample from which red blood cells have been removed as a specific component in blood, a known density gradient centrifugation method can be used.

  The separation medium used in the density gradient centrifugation is not particularly limited as long as it can be adjusted to specific gravity, osmotic pressure and pH without destroying cells. For example, commercially available Ficoll (registered trademark) and Percoll ( Registered trademark) or a sucrose solution.

  Then, by setting the specific gravity of the separation medium to be smaller than that of red blood cells and larger than that of white blood cells, the blood-derived sample is separated into at least two layers: a layer containing many red blood cells and a layer containing many cells other than red blood cells. It is possible to remove red blood cells from a blood-derived sample by taking out only the layer containing many cells other than red blood cells after density gradient centrifugation.

  In addition, two or more kinds having different specific gravities (for example, specific gravities of 1.077 and 1.119) may be used in combination as the separation medium. In this case, “cells other than erythrocytes can be further separated. Cells can be further eliminated.

Further, in the present specification, “the remaining blood-derived sample in which platelets are trapped” means a sample obtained by removing platelets from the above-mentioned blood-derived sample, but strictly excluding all platelets. It does not have to be, and includes those in which some platelets remain.
<First Embodiment>
As shown in FIGS. 1 and 2, the cell separation device 10 according to the first exemplary embodiment of the present invention includes a plate-shaped main body 12, and the upper surface of the main body 12 has one side. (Right side in FIG. 1) The platelet trap region 14 is provided in a biased manner.

  The platelet trap region 14 is formed by immobilizing a substance that interacts with platelets on the main body 12.

  The substance that interacts with platelets is preferably a substance that interacts with the membrane glycoprotein expressed on the platelet membrane, more preferably an antibody that interacts with the membrane glycoprotein. .

  Examples of such membrane glycoproteins include those listed in Table 1 below.

  Further, substances and antibodies that interact with the exemplified membrane glycoprotein are as described in Table 1.

ここで表１に例示した膜糖タンパク質と相互作用する物質および抗体について、血小板とＣＴＣの複合体が、この血小板トラップ領域１４でトラップされないようにするために、アフィニティー（親和性）の弱いものを選択することが好ましい。

Here, with respect to substances and antibodies that interact with the membrane glycoproteins exemplified in Table 1, in order to prevent the platelet-CTC complex from being trapped in the platelet trap region 14, those having weak affinity (affinity) are used. It is preferable to select.

A dissociation constant (K _D ) can be used as an index of affinity, and the dissociation constant (K _D ) is preferably in the range of 10 ⁻⁷ to 10 ⁻⁶ M. Further, among substances that interact with membrane glycoprotein, it is preferable to target GP IV-IX complex, and therefore, among antibodies that interact with membrane glycoprotein, anti-CD42b antibody should be selected. Is preferred. Among them, it is preferable to select an antibody clone having a dissociation constant (K _D ) of about 10 ⁻⁷ to 10 ⁻⁶ M.

  The substance that interacts with the membrane glycoprotein in the platelet trap region 14 is preferably one type, but a plurality of types may be used in some cases.

  On the other hand, in a region other than the platelet trap region 14 on the upper surface of the main body 12, that is, in a region on the other side 42 adjacent to the platelet trap region 14, blood for trapping blood circulating cancer cells (CTC) and other cells. A circulating cancer cell (CTC) trap region 16 is provided.

  In the CTC trap region 16 of the main body 12, a plurality of grooves 18 are arranged in a line from the one side 40 toward the other side 42.

  The plurality of grooves 18 provided in the CTC trap region 16 are preferably set to have a groove width T in the range of 1 to 100 μm and a groove depth H in the range of 1 to 100 μm as shown in FIG. .

  If the size of the groove 18 is within such a range, it can be trapped by cells remaining in the blood-derived sample 30 after separation of platelets, that is, leukocytes, CTC, a complex of platelets and CTC, and the platelet trap region 14. A few cells such as platelets that did not exist can be reliably developed in the groove 18.

  Moreover, it is preferable that the groove | channel 18 of the said dimension sets the groove width T and the groove depth H to the substantially same dimension. By setting in this way, it is difficult for the cells that have once entered the groove 18 to move, and it is also possible to prevent the cells that have entered the groove 18 from blocking other cells.

  Further, the cross-sectional shape of the groove 18 is not particularly limited, but a V shape as shown in FIGS. 1 to 3 is preferable because cells can be arranged closely.

  In addition to the V shape, for example, as shown in FIG. 4 (a), only one side surface of the groove 18 is slanted, and the top and bottom are rounded as shown in FIG. 4 (b). Or a U-shaped groove as shown in FIG. 4C.

  Further, a slit (not shown) may be provided at the bottom of the groove 18 so that moisture other than cells in the blood-derived sample can be discharged. In addition, when providing a slit (not shown), it is preferable to set the size of the slit so that the fluidity of the cells is not impaired and to adjust the amount of water discharged.

  The groove 18 provided in the CTC trap region 16 of the main body 12 can be processed by a known fine processing technique, but for example, a commercially available prism sheet can be substituted. Usually, the prism sheet has a plurality of grooves of the same size arranged in parallel on the upper surface of a flat plate, and the groove width T and groove depth H of the grooves are also suitable for the preferred range of the present invention. Has been.

  The main body 12 may be prepared separately for the platelet trap region 14 and the CTC trap region 16 and later joined using an adhesive or the like.

  The cell separation device 10 having such a structure is used in the platelet trap region 14 on the main body 12 from one side 40 toward the other side 42 in the direction of the arrow described in FIG. By supplying the sample 30, platelets in the blood-derived sample 30 are trapped in the platelet trap region 14.

  Note that the platelets trapped in the platelet trap region 14 do not have to be strictly all, and it is sufficient that an amount of CTC not trapped in the platelets is trapped at the time of subsequent CTC detection. Is.

  The amount of platelets to be trapped depends on the storage period (elapsed time since blood collection) and amount of the blood-derived sample 30 to be supplied, and the density of the antibody immobilized on the platelet trap region 14. It is preferable to adjust. However, since the platelet size is larger than the antibody, the dependence on the platelet amount is larger than the antibody density.

When the blood-derived sample 30 immediately after blood collection is 1 ml of blood, the sample after density gradient centrifugation contains an average of about 4.0 × 10 ⁶ platelets. The size of platelets is about 3 μm. For example, when a 4 cm ² platelet trap region 14 is used on a normal slide glass, at least 1 × 10 ⁶ platelets can be trapped in that region. Therefore, in order to remove platelets from 1 ml of blood, it is preferable to use about 4 such glass slides. However, this is the case where the antibodies immobilized on the platelet trap region 14 maintain the reactivity and are present most closely. In addition, blood that has passed from the time of blood collection can be processed with a smaller platelet trap region 14 because platelets have decreased.

  Next, a cell separation method using the above-described cell separation device 10 will be described.

  First, a cell separation device 10 having a plate-like body as shown in FIGS. 1 and 2 having the above-described configuration is prepared.

  Next, a blood-derived sample 30 is supplied onto the platelet trap region 14 from the one side 40 where the platelet trap region 14 of the cell separation device 10 is formed to the other side 42. The interacting substance traps platelets in the blood-derived sample 30.

  Here, in order for the interaction with platelets to sufficiently work in the platelet trap region 14, first, the blood-derived sample 30 is blood-derived using a flow rate adjusting means (not shown) so as to remain in the platelet trap region 14 for a certain period of time. It is preferable to control the flow rate of the sample 30.

  The method for supplying the blood-derived sample 30 to the platelet trap region 14 is not particularly limited. For example, after the cell separation device 10 is placed on a flat surface, the flow rate adjusting means (not shown) is used. The blood-derived sample 30 can be supplied from the one side 40 toward the other side 42 using a syringe.

  Alternatively, the cell separation device 10 may be slightly inclined, and the blood-derived sample 30 may be developed in the platelet trap region 14 by flowing the blood-derived sample 30 from the one side 40 of the main body 12.

  In short, any method may be used as long as each cell in the blood-derived sample 30 is moved using a gentle flow or its own weight.

  Next, after sufficiently interacting with platelets in the platelet trap region 14, the blood-derived sample 30 is supplied at a high flow rate of 100 to 1000 μl / sec.

  Thereby, even if the complex of platelets and CTC is trapped by the substance that interacts with platelets in the platelet trap region 14, the affinity of the substance that interacts with platelets as described above can be obtained. Since it is weak, the trap can be released at such a relatively fast flow rate.

  Therefore, in the platelet trap region 14, platelets in the blood-derived sample 30 are trapped by a substance that interacts with platelets, whereby the platelets can be separated from the blood-derived sample 30.

  In the cell separation device 10 according to the first embodiment described above, the blood circulating cancer cell (CTC) trap region 16 is provided adjacent to the platelet trap region 14, so that the remaining blood from which platelets have been separated is provided. The derived sample 30 is continuously flowed into the CTC trap region 16 located on the downstream side (the other side 42) of the platelet trap region 14. Here, the “remaining blood-derived sample 30 from which platelets have been separated” does not have to be strictly the remaining blood-derived sample 30 from which all of the platelets have been removed, and in a state where many platelets have been removed. If there is, it is good.

  Therefore, in the CTC trap region 16, cells containing CTC in the remaining blood-derived sample 30 from which platelets have been separated are expanded in the groove 18 in an aligned state.

Then, if the cells aligned in the groove 18 of the CTC trap region 16 are subsequently detected using, for example, an optical detection means (not shown), the presence / absence or number of CTCs, the state of the cells, etc. are accurately determined. It can be detected well.
<Second Embodiment>
FIG. 5 is a view showing a second embodiment of the cell separation device 10 of the present invention.

  The cell separation device 10 shown in FIG. 5 has a main body 12 having a bottomed tubular shape, which is different from the plate-like one as in the first embodiment.

  In such a cell separation device 10, a platelet trap region 14 is provided circumferentially on a part of the inner peripheral surface of the main body 12.

  The position of the platelet trap region 14 may be set according to the amount of the blood-derived sample 30 described later, and is preferably provided at a position where the platelet trap region 14 is immersed in the blood-derived sample 30.

  When performing cell separation using such a cell separation device 10, first, a blood-derived sample 30 is placed in the bottomed tubular main body 12 until the platelet trap region 14 is hidden, and in this state. The bottomed tubular main body 12 is rotated for a predetermined time to stir the internal blood-derived sample 30.

  Then, the platelets in the blood-derived sample 30 are trapped in the circumferentially provided platelet trap region 14.

  The blood-derived sample 30 remaining in the bottomed tubular main body 12 is in a state in which platelets are trapped in the platelet trap region 14 and hardly enter, and the remaining blood-derived sample 30 is separately planarized. By expanding, the remaining cells including CTC are expanded into a single layer.

By detecting using an optical detection means (not shown) in this state, it is possible to accurately detect the presence / absence and number of CTCs or the state of cells.
<Third Embodiment>
FIG. 6 is a view showing a third embodiment of the cell separation device 10 of the present invention.

  The cell separation device 10 shown in FIG. 6 has a well shape in which the main body 12 has a plurality of recesses 20, and is different from the plate-like shape as in the first embodiment.

  In such a cell separation device 10, the platelet trap region 14 is provided on the upper surface of the main body 12 excluding the recess 20.

  The diameter of the recess 20 is preferably in the range of 1 to 500 μm. By setting the diameter in such a range, the blood-derived sample 30 excluding platelets can surely fall into the recess 20 and be recovered. It has become so.

  And when performing cell separation using such a cell separation device 10, the sample 30 derived from blood is first inject | poured into the well-shaped main-body part 12 which has the recessed part 20, and the main-body part 12 is made into this state. The sample 30 derived from the blood is agitated for a predetermined period of time.

  Thereby, platelets in the blood-derived sample 30 are trapped in the platelet trap region 14 provided on the upper surface of the main body 12 excluding the recess 20.

  At this time, the blood-derived sample 30 excluding platelets falls into the recess 20 and is collected.

  When the blood-derived sample 30 excluding the platelets that have fallen into the concave portion 20 and is collected is developed in a flat plane as in the case of the second embodiment, the remaining cells including CTC are spread into a single layer. It will be.

  By detecting using an optical detection means (not shown) in this state, it is possible to accurately detect the presence or number of blood circulating cancer cells (CTC) or the state of the cells.

  In addition, when a small amount of the blood-derived sample 30 excluding platelets falling into the recess 20 and cells including CTC spread on a single layer on the bottom surface of the recess 20, detection means ( By detecting cells by adjusting the focal position (not shown), it is possible to accurately detect the presence or number of blood circulating cancer cells (CTC), the state of the cells, or the like. When such a method is used, it is not necessary to separately develop the blood-derived sample 30 excluding platelets in a flat shape, so that no CTC detection loss occurs and the CTC detection accuracy can be improved. .

  The cell separation device and the cell separation method using the same according to the present invention have been described above. However, the present invention is not limited to the above-described form and method. For example, the cell separation device according to the embodiment shown in FIG. The platelet trap region has a flat and flat shape, and various modifications can be made without departing from the object of the present invention, such as the formation of fine grooves.

[Example 1]
In the cell separation device 10 of the present invention and the cell separation method using the same, various blood cells in peripheral blood obtained by removing platelets after removing red blood cells from peripheral blood collected from cancer patients and further separating platelets. In particular, CTC was detected.
<Blood-derived sample 30>
Blood whose blood coagulation was blocked using ethylenediaminetetraacetic acid (EDTA) or heparin sulfate was diluted with phosphate buffered saline (PBS) supplemented with bovine serum albumin (BSA). This was layered in a test tube containing a Ficoll density gradient medium in advance, and centrifuged at room temperature using a centrifuge.

  After centrifugation, the entire upper layer including the mononuclear cell layer was collected from the test tube, and the sample tube was rotated and mixed well. This was used as a blood-derived sample 30.

This blood-derived sample 30 is considered to contain mononuclear cells (monocytes, lymphocytes), platelets, and other rare cells (CTC, vascular endothelial cells, vascular endothelial precursor cells, stem cells, etc.).
<Cell separation device 10>
A cell separation device 10 composed of the plate-like main body portion 12 shown in FIG. 1 is prepared, and a substance that interacts with platelets is immobilized on one side 40 on the cell separation device 10, and the platelet trap region 14. Was provided.

  As a method for immobilizing a substance that interacts with platelets on the main body 12, a general physical adsorption method was used.

  As a substance that interacts with platelets, an antibody against platelet membrane glycoprotein (CD42b) (manufactured by Takara Bio Inc.) was used.

  Specifically, an anti-CD42b antibody diluted to 2 to 5 μg / ml with a sodium bicarbonate buffer (0.1 M, pH 9.6) is immersed in the platelet trap region 14 and is allowed to stand at room temperature for 3 hours (or overnight at 4 ° C.). Reacted.

  The anti-CD42b antibody that was not adsorbed on the platelet trap region 14 was collected, washed with a phosphate buffered saline containing 0.05% Tween (hereinafter also referred to as PBS), and further washed with PBS.

  After the washing, the cell separation device 10 was filled with 5% skim milk or 1% BSA, reacted at room temperature for 2 hours (or overnight at 4 ° C.), and blocked.

  After the blocking, it was washed again with PBS. Note that, except for the platelet trap region 14, masking was performed so that the above reaction did not occur.

The method for immobilizing the antibody that interacts with the membrane glycoprotein may be based on a chemical bond using a solid support such as a polymer, regardless of this method.
<Development and separation of cells>
For the development of the blood-derived sample 30 on the cell separation device 10, a fluid development method was used, as in a method using a general microfluidic device and a laminar flow chamber.

  The fluid such as the blood-derived sample 30 was introduced and discharged using an opening of the laminar flow chamber and under the control of a pump capable of setting the flow rate.

  Here, the blood-derived sample 30 or PBS may be reciprocated.

  First, the blood-derived sample 30 is introduced into the platelet trapping region 14 at a slow flow rate of 10 to 100 μl / sec so that the platelets can reliably bind to the anti-CD42b antibody that interacts with the membrane glycoprotein and left for a while. Then, after the reaction was completed, PBS was sufficiently poured at a similar flow rate for washing.

Next, in order to dissociate the complex of CTC and platelets, which is weakly bound to the anti-CD42b antibody, and other non-specifically bound substances, the PBS is placed in the platelet trap region 14 at a high flow rate of 500 to 1000 μl / sec. Shed.
<Cell detection>
In order to identify the cells on the cell separation device 10 by optical detection, fluorescent immunostaining was performed.

  First, to retain intracellular and cell surface antigens, a 4% paraformaldehyde (manufactured by Wako Pure Chemical Industries) solution was reacted for 15 minutes at room temperature, washed with a sufficient amount of PBS, and then the cell membrane. As a permeation treatment, the reaction was performed with PBS containing 0.1% Tween for 30 minutes.

  Next, PBS containing anti-CK antibody (Micromet) labeled with Alexa Floor 647 (Invitrogen) for CTC staining, and anti-CD45 antibody (Santa Cruz Biotechnology, Inc.) labeled with Alexa Floor 488 for leukocyte staining Was allowed to react for 40 minutes. Among these, for the last 5 minutes, a DAPI (manufactured by Dojin Chemical) solution was added for cell nucleus staining.

  After completion of the reaction, the cell separation device 10 was washed several times with a sufficient amount of PBS to remove excess antibody and fluorescent dye.

  For the above-described fluorescence detection, commonly used detection means such as a fluorescence microscope and an image scanner were used.

  A region other than the platelet trap region 14 (CTC trap region 16) of the cell separation device 10 was irradiated with a He—Ne laser (excitation wavelength: 633 nm) for exciting Alexa Floor 647, and CK was detected.

  At the same time or next, Ar laser (excitation wavelength: 488 nm) for exciting Alexa Floor 488 was similarly irradiated to detect CD45, and DAPI was UV-excited using a mercury lamp to detect cell nuclei.

  From signals obtained by these irradiations, a signal in which CK and DAPI were detected and CD45 was not detected was identified as CTC.

  For confirmation, no signal was observed when the above-described laser was irradiated to the platelet trap region 14.

  Thereby, if the cell separation device 10 and the cell separation method using the same according to Embodiment 1 of the present invention are used, platelets can be accurately separated from a plurality of types of cells contained in the blood-derived sample 30. It was confirmed that

  Therefore, it was confirmed that CTC in the blood-derived sample 30 from which platelets were separated could be detected with high accuracy.

  In Example 1 described above, CTC was detected in peripheral blood. However, if the cell separation device 10 of the present invention and the cell separation method using the same are used, platelets in the blood-derived sample 30 are detected. Since it can be separated with high accuracy, it can also be used to detect rare cells such as vascular endothelial cells, vascular endothelial precursor cells, and various stem cells in peripheral blood.

DESCRIPTION OF SYMBOLS 10 ... Cell separation device 12 ... Main-body part 14 ... Platelet trap area | region 16 ... Blood circulation cancer cell (CTC) trap area | region 18 ... Groove 20 ... Recessed part 30 ... Blood origin Sample 40 ... One side 42 ... The other side T ... Groove width H ... Groove depth E ... Diameter 100 ... Test tube 200 ... Blood-derived sample 202 ... Plasma 204 ... Platelet 206 ... White blood cell 208 ... Red blood cell 300 ... Blood circulating cancer cells (CTC)

Claims (28)

A cell separation device for separating desired cells from a plurality of types of cells contained in a blood-derived sample,
The cell separation device includes a main body,
The main body is provided with a platelet trap region for trapping at least platelets in the blood-derived sample,
The cell separation device, wherein the platelet trap region is formed by immobilizing a substance that interacts with the platelet on the main body. The main body is plate-shaped,
The cell separation device according to claim 1, wherein the platelet trap region is provided on a part of the upper surface of the plate-shaped main body.   The cell separation device according to claim 2, wherein the platelet trap region is provided to be biased to one side of the upper surface of the main body. The platelet trap region provided on a part of the upper surface of the main body portion,
The cell separation device according to claim 2 or 3, wherein the cell separation device has a flat planar shape.   The blood circulation cancer cell (CTC) trap region for trapping blood circulation cancer cells (CTC) and other cells is further provided on the upper surface of the main body. The cell separation device according to any one of the above. In the blood circulation cancer cell (CTC) trap region,
The cell separation device according to claim 5, wherein a plurality of grooves are arranged in a line from one side of the main body part to the other side.   The cell separation device according to claim 6, wherein the groove width of the groove is in the range of 1 to 100 μm and the groove depth is in the range of 1 to 100 μm.   The cell separation device according to claim 6 or 7, wherein the groove has a V-shaped cross section. The main body is a bottomed tubular,
2. The cell separation device according to claim 1, wherein the platelet trap region is provided circumferentially on a part of an inner peripheral surface of the bottomed tubular main body portion. The main body has a well shape having a plurality of recesses,
The cell separation device according to claim 1, wherein the platelet trap region is provided on an upper surface of the well-shaped main body portion excluding the concave portion.   The diameter of the said recessed part exists in the range of 1-500 micrometers, The cell separation device of Claim 10 characterized by the above-mentioned.   The cell separation device according to any one of claims 1 to 11, wherein the blood-derived sample is a sample after red blood cell separation processing has been performed in advance.   The cell separation device according to claim 12, wherein the separation process of the red blood cells is performed by density gradient centrifugation. The specific gravity of the separation medium used in the density gradient centrifugation method is
14. The cell separation device according to claim 13, which is smaller than red blood cells and larger than white blood cells.   The cell separation device according to any one of claims 1 to 14, wherein the substance that interacts with platelets is a substance that interacts with a membrane glycoprotein expressed on a cell membrane of the platelets.   16. The cell separation device according to claim 15, wherein the substance that interacts with the membrane glycoprotein is an antibody that interacts with the membrane glycoprotein. The cell separation device according to claim 15 or 16, wherein the substance that interacts with the membrane glycoprotein is a substance having a dissociation constant (K _D ) in the range of 10 ^-7 to 10 ^-6 M. A cell separation method for separating desired cells from a plurality of types of cells contained in a blood-derived sample,
The cell separation method comprises:
A cell separation device having a platelet trap region in which a substance that interacts with platelets in the blood-derived sample is immobilized is prepared on the main body, and the blood-derived device is placed on the platelet trap region of the cell separation device A step of trapping the platelets in the blood-derived sample in the platelet trap region;
A cell separation method characterized by comprising: In the step of trapping the platelets,
When supplying the blood-derived sample on the platelet trap region, the flow rate of the blood-derived sample is adjusted using a flow rate adjusting means so that the blood-derived sample remains on the platelet trap region. The cell separation method according to claim 18. In the cell separation device prepared in the step of trapping the platelets,
A blood circulation cancer cell (CTC) trap region for trapping blood circulation cancer cells (CTC) and other cells is further provided on the upper surface of the main body.
Following the step of trapping the platelets,
Supplying the blood-derived sample to the blood circulation cancer cell (CTC) trap region, and trapping the blood circulation cancer cell (CTC) and other cells in the blood circulation cancer cell (CTC) trap region When,
The cell separation method according to claim 18 or 19, characterized by comprising:   The cell separation method according to claim 19 or 20, wherein a flow rate of the blood-derived sample is in a range of 100 to 1000 µl / sec. Following the step of trapping the platelets,
Developing the remaining blood-derived sample in which the platelets are trapped in a planar shape;
The cell separation method according to claim 18, comprising:   The cell separation method according to any one of claims 18 to 22, wherein the blood-derived sample is a sample after red blood cell separation processing has been performed in advance.   The cell separation method according to claim 23, wherein the red blood cell separation process is performed by density gradient centrifugation. The specific gravity of the separation medium used in the density gradient centrifugation method is
The cell separation method according to claim 24, which is smaller than red blood cells and larger than white blood cells.   The cell separation method according to any one of claims 18 to 25, wherein the substance that interacts with platelets is a substance that interacts with a membrane glycoprotein expressed on a cell membrane of the platelets.   26. The cell separation method according to claim 25, wherein the substance that interacts with the membrane glycoprotein is an antibody that interacts with the membrane glycoprotein. 28. The cell separation method according to claim 26 or 27, wherein the substance that interacts with the membrane glycoprotein is a substance having a dissociation constant (K _D ) in the range of 10 ⁻⁷ to 10 ⁻⁶ M.

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