JP2012000065A - Cell arrangement system and cell arrangement method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a cell arrangement system capable of arranging a large quantity of cells without being aggregated.SOLUTION: The cell arrangement system includes a main channel in which a cell suspension containing cells in blood flows; a subordinate channel whose one end is connected to a side surface of the main channel; a first filter disposed between the main channel and the subordinate channel and having a filter surface with openings smaller than the diameter of cells to be arranged; a first feed pump for feeding a liquid in a forward direction of the main channel; and a second feed pump for feeding the liquid in the opposite direction from the forward direction of the main channel. The cell suspension is fed in the forward direction of the main channel by the first feed pump to permeate over the first filter, and the permeated cell suspension is fed in the opposite direction of the main channel by the second feed pump to permeate over the first filter again, so that the cells to be arranged are arranged in the first filter in the cell arrangement system.

Description

  The present application relates to a cell arrangement system and a cell arrangement method for arranging cells in order to detect cells in blood, particularly circulating tumor cells (CTC).

  Tumor cells are present in the blood of cancer patients. By detecting the presence or absence of tumor cells in the circulating blood, it is possible to determine whether or not the patient is afflicted with cancer or the effect of treatment on a cancer patient.

  The concentration of tumor cells is very low and exists in only a few low concentrations in 107-108 cells. For example, there are about several tumor cells contained in a 10 ml sample of blood.

  In the method disclosed in Patent Document 1, rare particles such as circulating tumor cells are rarely used by using magnetic particles coupled to biologically specific ligands that react specifically with rare cells. The presence and number of rare cells are detected by separating the cells from the blood, labeling the separated rare cells, and observing the labeled rare cells.

  Patent Document 2 describes a method of isolating cells through blood containing circulating tumor cells in a membrane filter.

  In Patent Document 3 and Patent Document 4, a micromesh having a plurality of cylindrical or mortar-shaped fine through-holes is disposed in a part of the microchannel in order to separate and capture from a large number of cells at one cell level, A method is disclosed in which a sample containing cells is injected into a fine channel and the lower part of the micromesh is sucked with a predetermined suction pressure.

JP-T-2002-503814 Special table 2008-538509 gazette JP 2007-89566 A International Publication No. 2009/016842

  In Patent Document 1, in the process of extracting or limiting rare cells to be observed to rare cells, the rare cells may be missed, and as a result, the detection sensitivity may decrease.

  In order to prevent missing, all the rare cells and white blood cells to be observed should be observed. In that case, the cells must be expanded so that they do not overlap each other. However, simply expanding it requires a very wide observation area, and in this case, the size of the detection device increases, which is not practical.

  Further, in Patent Documents 2 to 4, cells are retained by filtration through a filter. However, since cells tend to aggregate, it is structurally possible to develop a large number of aggregated cells, align them, and optically observe them. difficult.

  In view of such problems, the present invention has an object to provide a cell arrangement system and a cell arrangement method capable of arranging a large amount of cells without aggregating them.

  The above object is achieved by the invention described below.

1. A cell arrangement system for arranging cells in blood,
A main channel for flowing a cell suspension containing cells in the blood;
A sub-flow path having one end connected to a side surface of the main flow path;
A first filter provided between the main flow path and the sub flow path and having a filter surface having an opening smaller than the diameter of cells to be arranged;
A liquid feeding mechanism for feeding liquid in the forward direction and the reverse direction of the main flow path;
Have
By the liquid feeding mechanism, the cell suspension is fed in the forward direction of the main channel and passed over the first filter, and then the cell suspension is fed in the reverse direction of the main channel and the first channel is sent. The cell arrangement system, wherein the cells to be arranged are arranged on the first filter by passing the filter again over one filter.

  2. The cell suspension is reciprocated a plurality of times on the first filter by repeating liquid feeding in the forward direction of the main flow path and liquid feeding in the reverse direction of the main flow path by the liquid feeding mechanism. 2. The cell array system according to 1 above.

3. The liquid feeding mechanism has a first liquid feeding pump for feeding in the forward direction of the main flow path and a second liquid feeding pump for feeding in the reverse direction of the main flow path,
A cell suspension is fed in the forward direction of the main flow path by the first liquid feed pump and passed over the first filter, and the passed cell suspension is passed through the main flow path by the second liquid feed pump. The cell arrangement system according to 1 or 2, wherein the cells to be arranged are arranged on the first filter by sending the solution in the opposite direction and passing the first filter again.

4). The liquid feeding mechanism includes a first liquid feeding pump, and a path switching unit that switches a liquid feeding path by the first liquid feeding pump to a first path and a second path,
The cell suspension is fed in the forward direction of the main flow path by the first liquid feed pump having the liquid feed path as the first path by the path switching means, and passed over the first filter,
The passed cell suspension is fed in the direction opposite to the main flow path by the first liquid feed pump whose second path is the second path by the path switching means, and again passes over the first filter. 3. The cell arrangement system according to 1 or 2, wherein the cells to be arranged are arranged on the first filter.

5). The liquid feeding mechanism has a third liquid feeding pump connected to the downstream side of the sub-flow path,
5. The cell array system according to any one of 1 to 4, wherein the third flow pump is used to create a negative pressure in the subchannel.

6). The liquid feeding mechanism includes a first liquid feeding pump capable of switching a liquid feeding direction between a forward direction of the main flow path and a reverse direction of the main flow path, and a third liquid feed pump connected to the downstream side of the sub flow path. Have
In a state in which the inside of the auxiliary flow path by the third liquid feeding pump is set to a negative pressure,
A cell suspension is fed in the forward direction of the main flow path by the first liquid feed pump and passed over the first filter, and the passed cell suspension is passed through the main flow path by the first liquid feed pump. The cell arrangement system according to 1 or 2, wherein the cells to be arranged are arranged on the first filter by sending the solution in the opposite direction and passing the first filter again.

  7). The main channel, which is disposed downstream of the first filter in the forward direction of the main channel, has a filter surface formed in a direction intersecting the channel axis direction, and is smaller than the diameter of the cells to be arranged The cell arrangement system according to any one of 1 to 6, further comprising a second filter having a small aperture.

8). The other end different from the one end of the sub flow path is connected to the main flow path,
8. The cell suspension sent into the sub-flow path by a third liquid feed pump is sent from the other end to the main flow path, as described in any one of 5 to 7 above. Cell array system.

  9. Any one of 5 to 7 above, wherein the third flow pump is used to make the inside of the sub-flow path positive and negative, and by alternately repeating this, vibration is applied to the first filter. The cell arrangement system according to Item.

10. A filter surface having a main channel, a sub channel whose one end is connected to a side surface of the main channel, and an opening smaller than the diameter of the cell to be arranged, which is provided between the main channel and the sub channel. A cell arrangement method for arranging cells on the first filter of a chip structure having a formed first filter,
A first step of passing the cell suspension over the first filter by feeding a cell suspension containing cells in blood in the forward direction of the main flow path;
And a second step of allowing the liquid to flow again in the reverse direction of the main flow path so as to pass through the first filter again.

  11. 11. The cell arrangement method according to 10 above, wherein the first step and the second step are repeated.

12 A pump for changing the pressure inside the sub-flow path is connected;
In the above 10 or 11, characterized by having a vibration step of applying vibration to the first filter by making the pressure inside the sub-flow path positive and negative by the pump and alternately repeating this. The cell array method described.

  According to the present invention, it is possible to arrange a large amount of cells without aggregating them by reciprocating the cell suspension on the filter.

It is a figure which shows the structure of the outline of the cell arrangement | sequence system which concerns on 1st Embodiment. 2A is a cross-sectional view of the chip structure 10 in the side surface direction, and FIG. 2B is a cross-sectional view in the upward direction. 3A is a cross-sectional view of the first filter 101, and FIG. 3B is a top view. It is a figure which shows the state in which the white blood cell c is trapped by the hole h. FIG. 3 is a diagram illustrating an arrangement method in the chip structure 10. It is a figure which shows the schematic structure of the cell arrangement | sequence system which concerns on 2nd Embodiment. It is a figure which shows the schematic structure of the cell arrangement | sequence system which concerns on 3rd Embodiment. FIG. 8A is a cross-sectional view in the side surface direction of the chip structure 10 according to the fourth embodiment, and FIG. 8B is a cross-sectional view in the upward direction. It is a figure explaining the arrangement | sequence method in the chip structure 10 which concerns on 4th Embodiment. It is a figure which shows the schematic structure of the cell arrangement | sequence system which concerns on 5th Embodiment. 11A is a cross-sectional view of the chip structure 10 in the side surface direction, and FIG. 11B is a cross-sectional view in the upward direction. It is a time chart which shows operation | movement of each pump in the cell arrangement | sequence system concerning a modification.

  Although the present invention will be described based on an embodiment, the present invention is not limited to the embodiment.

  FIG. 1 is a diagram illustrating a schematic configuration of a cell arrangement system 1A according to the first embodiment and a cell detection system including the same.

  The cell detection system shown in the figure includes an imaging unit 25, an image analysis unit 20, and a cell arrangement system 1A. The cell array system 1A includes a first liquid pump p1, a second liquid pump p2, a third liquid pump p3, a chip structure 10, a control unit 15, a supply tank 16, a valve b1, and a discharge tank 17. . The control unit 15 controls each pump and the valve b1. The liquid feeding mechanism in the first embodiment includes at least a first liquid feeding pump p1 that applies positive pressure, a second liquid feeding pump p2 that applies positive pressure, and further includes a third liquid feeding pump p3. You may have.

  The blood and other cell suspensions supplied to the supply tank 16 are supplied to the chip structure 10 via the flow path r1 by the first liquid feeding pump p1. A first filter 101 is provided inside the chip structure 10, and blood cells and other cells contained in the cell suspension are arranged on the first filter 101. The arrangement method will be described later.

  The cells arranged in the first filter 101 are imaged by the imaging unit 25, and the obtained image data is subjected to image analysis by the image analysis unit 20. The term “arrangement” here means that all cells are ideally arranged in a single layer and in a high density. By arranging each cell in a high density without overlapping each other, the shape of all the cells is photographed. Can be confirmed. For image analysis, for example, if the purpose is to select rare cells contained in blood, the individual cells arranged in the first filter 101 are extracted. Based on the shape of the extracted cells, white blood cells, tumor cells and other rare cells are selected to detect the presence or number of rare cells.

  FIG. 2 is an enlarged cross-sectional view of the chip structure 10. 2A is a side sectional view, and FIG. 2B is an upward sectional view. The chip structure 10 includes a main channel 102, a sub channel 103, and a first filter 101. One end of the main flow path 102 is connected to the first liquid feed pump p1 via the flow path r1, and the other end is connected to the second liquid feed pump p2 via the flow path r2. One end of the sub channel 103 is connected to the side surface of the main channel 102, and the other end is connected to the channel r3. As shown by the arrows in the figure, the feeding direction by the first liquid feeding pump p1 is the forward direction fd, and the feeding direction by the second liquid feeding pump p2 is the opposite reverse direction rd.

The size of the first filter 101 is, for example, 0.01 mm ² of 100 mm □, and the channel cross-sectional area of the main channel 102 is 10 mm ² (width 100 mm, height 0.1 mm). The amount of the cell suspension bs to be sent is 2000 μl, and the flow rate in each direction is 1-1000 μl / min, more preferably 10-100 μl / min.

  The first filter 101 is provided on the side surface of the main channel 102, the filter surface is parallel to the channel axis direction of the main channel 102, and the streamline and channel axis of the sub-channel 103. It spreads in a direction orthogonal to the direction. In addition, the imaging unit 25 is disposed immediately above the first filter 101 of the chip structure 10 shown in FIG. The housing of the chip structure 10 uses a light-transmitting material (for example, alkali glass, quartz glass, transparent plastics) at least above the first filter 101 in order to perform optical imaging by the imaging unit 25. Light is transmitted.

  FIG. 3 is a schematic diagram illustrating the structure of the first filter 101. 3A is a cross-sectional view of the first filter 101, and FIG. 3B is a top view. FIG. 4 is a diagram corresponding to FIG. 3A, and shows a state in which the white blood cells c are trapped in the holes h by the first filter 101.

In the first filter 101 shown in these drawings, a large number of holes h are arranged over an area of, for example, 0.01 m 2 with a predetermined hole density. The pore density for example, ^{1 × 10 11 / m 2 ~6} × 10 12 / m 2. Each hole h is a through hole, and the diameter of the hole h is smaller than the diameters of the white blood cell c and the rare cell, for example, 0.5 μm. In the embodiment shown in FIG. 3, the through holes of the filter are equally arranged in the plane direction, but the present invention is not particularly limited thereto, and may be randomly arranged. For example, sintered metal mesh filters (Fuji Filter Industry Co., Ltd.), Nuclepore membrane (sales company of GE Healthcare Japan Co., Ltd.).

  FIG. 5 is a diagram for explaining the arrangement method in the chip structure 10 according to the present embodiment, which is displayed in time series from FIG. 5A to FIG. 5E.

  FIG. 5A to FIG. 5C are diagrams showing the first step. In the first step, the cell suspension bs is fed in the forward direction fd (see FIG. 5A) and passed through the first filter 101.

  FIG. 5A shows a state in which the cell suspension bs supplied from the supply tank 16 is supplied to the main channel 102 of the chip structure 10 via the channel r1 by the first solution pump p1. ing.

  In each figure, there is a gas such as air or an inert gas before and after the cell suspension bs, and a liquid feeding pressure is applied from each pump to the cell suspension bs via the gas. . In addition, the cell suspension used here is a pre-treated blood sample collected from a subject. As pretreatment, a stabilizer is added to the blood, diluted to a predetermined concentration, or red blood cells and platelets are removed. For example, the collected blood is mixed with a stabilizer, and then centrifuged at 1500 rpm for 5 minutes, and then the supernatant is removed and the remaining precipitate is used. The cell suspension bs contains at least white blood cells c (in some cases, white blood cells and rare cells).

  FIG. 5B shows a state where the cell suspension bs passes through the first filter 101. Since the sub flow channel 103 is provided below the main flow channel 102, a part of the cell suspension bs is branched by gravity and flows into the sub flow channel 103. At this time, since the diameter of the hole h of the first filter 101 is smaller than the diameter of the white blood cell c, the white blood cell c is trapped (captured) by the first filter 101.

  FIG. 5C shows a state in which the cell suspension bs fed from the upstream side is branched and sent to the main channel 102 and the sub channel 103 on the downstream side of the first filter 101. .

  In the state shown in FIG. 5C, the ratio R (R = M3 / M2) of the liquid amount M3 flowing through the sub-flow channel 103 to the liquid amount M2 flowing through the main flow channel 102 of the cell suspension bs is 2% to 50%. Is set. The ratio R varies depending on the fluidity of the cell suspension bs and the resistance of the first filter 101.

  The third liquid feed pump p3 is not always necessary, but is useful when the ratio R is to be increased or controlled. In the present embodiment, the ratio R is set within a predetermined range by setting the inside of the sub flow path 103 to a negative pressure with respect to the main flow path 102 by the third liquid feeding pump p3.

  FIG.5 (d) and FIG.5 (e) are figures which show a 2nd process. In the second step, the cell suspension bs in the main channel 102 that passes through the first filter 101 in the state shown in FIG. 5C is fed in the reverse direction rd by the second liquid feeding pump p2. In FIG.5 (c), the cell suspension bs which passed the 1st filter 101 at the 1st process is sent to the reverse direction by the 2nd liquid feeding pump p2, and is passing over the 1st filter 101 again. Indicates the state. In order to arrange the cells, it is desirable that one white blood cell c is trapped in each hole h of the first filter 101. This is because the white blood cells c can be arranged at a relatively high density without overlapping each other in such an aligned state.

  However, the white blood cell c on the first filter 101 trapped in the first step is not only the white blood cell c trapped in the hole h, but also the white blood cell present or trapped between these holes h. There are also white blood cells c that overlap and c aggregate. By passing the cell suspension bs again as shown in FIG. 5D to FIG. 5E, the state of the white blood cells c other than these trapped white blood cells c can be changed. The trap rate of one filter 101 can be improved. The white blood cell c already trapped in the first step is relatively difficult to move because it is caught in the hole h, so that the trapped state is maintained. As described above, since the trap rate can be improved by performing the second step following the first step, it is possible to arrange the cells contained in the cell suspension.

  In addition, following the second step, the first step and the second step may be repeated again to perform the reciprocating motion of the cell suspension bs on the first filter 101 a plurality of times. The number of reciprocating motions can be appropriately selected according to the value of the ratio R and the amount of the supplied cell suspension bs. By reciprocating a plurality of times, the trap rate can be further improved, and many cells can be arranged at high density without overlapping, so that a high level arrangement can be performed.

  Further, the cells can be more accurately observed by fixing and staining the cells as described below. When the chip structure of the present invention is used for liquid exchange operations such as cell fixation and staining, the cells after the reaction are arranged, and the number of times of contact between the cells and the substance in the reaction solution is reduced by the reciprocating solution. Since it increases, it is possible to increase the reaction efficiency.

  As a cell fixing method, for example, formalin method, methanol method or the like is performed. As a cell staining method, for example, DAPI staining or Hoechst staining for staining DNA in the nucleus, or immunostaining using an antibody specific to the cell type is performed. As immunostaining, for example, leukocyte common antigen CD45 and tumor marker CK (cytokeratin) are performed. Liquids that are subject to liquid exchange include liquids such as PFA that immobilize cells, lysates or dilutions for diluting specimens, various reaction reagents and washing reagents for detecting specific cells, and fluorescent dyes for antibodies And the like (for example, water-soluble carbodiimide (EDC and the like), N-hydroxysuccinimide [NHS] and the like) and the like.

  Imaging of the cells arranged in the first filter 101 is performed by the imaging unit 25, and rare cells are detected by analyzing the obtained image data. It should be noted that, when photographing, observation may be performed after the liquid is fed to the downstream side in the forward direction of the main channel 102 or the sub channel 103 so that the fine suspension bs does not remain on the first filter 101. preferable.

[Second and third embodiments]
Next, second and third embodiments will be described. In the embodiments described below as different from the first embodiment, the same components are denoted by the same reference numerals, and the description will be replaced. In FIGS. 6 and 7, descriptions of the control unit 15, the imaging unit 25, and the image analysis unit 20 are omitted.

  FIG. 6 is a diagram showing a schematic configuration of a cell arrangement system 1B in the second embodiment. In the second embodiment, the first liquid feeding pump p1b is configured to feed a liquid or air in a protruding direction by applying a positive pressure to the inside of a connected pipe to a positive pressure. Conversely, by applying a negative pressure, it is possible to switch the liquid feeding in the suction direction to make the inside of the tube a negative pressure. The liquid feeding mechanism in the second embodiment includes at least a first liquid feeding pump p1b that applies positive and negative pressures and a third liquid feeding pump p3 that applies negative pressure.

  Also in the second embodiment, the same arrangement method as shown in FIG. 5 is executed. At this time, the cell suspension bs is fed in the forward direction fd and the reverse direction rd in the same figure by the first liquid feed pump p1b. Specifically, the feed in the forward direction fd in the first step is performed by applying a positive pressure by the first liquid pump p1b, and the feed in the reverse direction rd in the second step is negative by the pump. This is done by applying pressure. At this time, the third liquid feeding pump p3 is driven during the second step, or between the first step and the second step, and the pressure inside the sub-flow channel 103 is negative with respect to the main flow channel 102. It is preferable to keep it.

  FIG. 7 is a diagram showing a schematic configuration of a cell arrangement system 1C in the third embodiment. In the example shown in the figure, the flow path between the first liquid delivery pump p1c and the chip structure 10 is branched into a flow path r1 that is a first path and a flow path r2 that is a second path. The three-way valve b10 is provided at the branch portion. The three-way valve b10 functions as a path switching unit and can switch the liquid feeding path by the first liquid feeding pump p1c in either the flow path r1 or r2. The liquid feeding mechanism in the third embodiment includes at least a first liquid feeding pump p1c that applies positive pressure, a three-way valve, and other path switching means, and may further include a third liquid feeding pump p3. .

  Also in the third embodiment, the same arrangement method as shown in FIG. 5 is executed. At that time, the liquid feeding in the forward direction fd and the reverse direction rd of the cell suspension bs in the same figure is switched by the control unit 15 by controlling the three-way valve b10 to switch the liquid feeding direction by the first liquid feeding pump p1c. To do. Specifically, in the forward direction fd in the first step, the positive pressure applied by the first liquid pump p1c to the cell suspension bc is controlled from the flow path r1 side by controlling the three-way valve b10. In the second step, feeding in the reverse direction rd is performed so that a positive pressure is applied from the flow path r2.

  In the example shown in FIG. 7, the downstream side of the flow path r <b> 3 shows an open state, but the third liquid feeding pump p <b> 3 is connected in the same manner as in FIG. 6, and thereby, during the first and second steps, You may make it the inside of the subchannel 103 become a negative pressure.

  Also in the configuration of the second and third embodiments, the trap rate can be improved by performing the first step and the second step, as in the first embodiment, so that it is included in the cell suspension. Cells can be arranged. Moreover, you may make it repeat a 1st process and a 2nd process in multiple times. This makes it possible to further improve the trap rate.

[Fourth Embodiment]
8A and 8B are views for explaining the structure of the chip structure 10 according to the fourth embodiment. FIG. 8A is a side sectional view, and FIG. 8B is an upper sectional view. The configuration of the cell arrangement system according to the fourth embodiment can be applied to the cell arrangement systems of the first to third embodiments.

  As shown in the figure, in the chip structure 10 in the fourth embodiment, a second filter 106 is provided. The second filter 106 is disposed downstream of the first filter 101 in the forward direction fd inside the main flow path 102, and its filter surface is formed in a direction intersecting the flow path axial direction of the main flow path 102. ing. In the figure, an example is shown in which the filter surface is formed in a direction orthogonal to the channel axial direction of the main channel 102.

  The configuration of the second filter 106 is the same as that of the first filter 101, and a description thereof is omitted. Since the second filter 106 is also provided with many holes having a diameter smaller than the cell diameter, the white blood cells c can be trapped.

  In the figure, the main channel 102 is linear, and the first filter 101 parallel to the channel axis direction of the main channel 102 and the second filter 106 perpendicular to the channel axis direction are orthogonal to each other. However, the present invention is not limited to this, and the first filter 101 and the second filter 106 may be parallel to each other as a configuration in which the main channel 102 is bent downstream in the forward direction fd of the first filter 101. . In the configuration in which the main channel 102 bends downward on the downstream side in FIG. 8, white blood cells c are trapped on the upper side in the gravity direction of the second filter 106. Similar to the first filter 101, the second filter 106 can be used as an observation region by photographing.

  FIG. 9 is a diagram illustrating an arrangement method in the chip structure 10 according to the fourth embodiment. 9 (a) to 9 (d) in the figure correspond to FIGS. 5 (a) to 5 (d).

  Since the second filter 106 is provided in the fourth embodiment, when the cell suspension bs is fed in the forward direction fd in the first step shown in FIG. 9A to FIG. 9C. The white blood cell c is trapped by the second filter 106 (see FIG. 9C). In the second step shown in FIG. 9 (d), the white blood cells c trapped in the second filter 106 are released from the trapped state at a time at the start of liquid feeding by liquid feeding in the reverse direction rd. At the beginning of the flow of the turbid liquid bs, many open white blood cells c are contained in the cell suspension bs, and the white blood cells c can be arranged on the first filter 101 with higher density. .

  Further, as shown in FIG. 9 (d), the proportion of white blood cells c contained therein is high in the front of the cell suspension bs fed in the reverse direction rd in the second step and low in the rear as shown in FIG. 9 (d). In some cases, it will hardly be included. Therefore, as shown in FIG. 9 (e), before all the cell suspension bs passes, the pump to be moved can be switched to the first liquid feeding pump p1 to reverse the feeding direction. Therefore, it is possible to arrange the white blood cells c on the first filter 101 in a shorter time.

[Fifth Embodiment]
10 and 11 are diagrams showing a schematic configuration of a cell arrangement system 1E according to the fifth embodiment. In the figure, the same components as those in the embodiment shown in FIGS. In the embodiment shown in the figure, one end of the sub flow path 103b is connected to the main flow path 102, and the other end is connected to the main flow path 102 via the valve b2 and the flow path r1.

  The cell suspension bs sent to the sub flow channel 103 is circulated from the other end side of the sub flow channel 103 to the main flow channel 102. By doing so, the cell suspension sent to the sub-flow channel 103 can be reused, so that the cell suspension flowing in the main flow channel 102 does not gradually decrease depending on the number of reciprocations, and is always the same. There is an effect of reciprocating in the state. This is particularly effective when the ratio R of the liquid amount M3 flowing through the sub-flow channel to the liquid amount M2 flowing through the main flow channel 102 of the cell suspension bs is increased.

[Modification]
FIG. 12 is a time chart showing the operation of each pump in the cell arrangement system according to the modification of the present embodiment. In the modification, the vibration step is performed after the first step and the second step are performed and the cell suspension is fed. In the time chart shown in the figure, the vibration process is performed after the first process and the second process are performed once, but the vibration process is performed after the first process and the second process are performed a plurality of times. Alternatively, the first step and the second step may be performed after the vibration step. Further, the timing of performing the vibration process is as follows. The cell suspension bs is passing through the first filter 101 as shown in FIG. 5B and FIG. The vibration feeding process may be executed by interrupting the liquid feeding by the two liquid feeding pump p2.

  In the vibration step, the internal pressure of the sub-flow channel 103 is changed to a positive pressure by sequentially switching the pressure application direction by the third liquid feeding pump p3 from the forward direction as shown in FIG. A negative pressure is applied to vibrate the white blood cell c existing on the first filter 101 or the cell suspension bs containing the white blood cell c. The frequency for changing the pressure application direction of the third liquid feeding pump p3 can be appropriately selected.

  The effect of separating the leukocyte cells c that overlap and aggregate with the leukocyte cells c trapped in the holes h on the first filter 101 by applying vibration to the leukocyte cells c or the cell suspension bs containing the leukocyte cells c. Can be expected. As a result, the white blood cells c can be arranged so as not to overlap each other.

1A, 1B, 1C, 1E Cell array system 15 Control unit 16 Supply tank 17 Discharge tank 20 Image analysis unit 25 Imaging unit p1, p1b, p1c First liquid pump p2 Second liquid pump p3 Third liquid pump b1, b2 valve r1, r2, r3 flow path 10 chip structure 101 first filter 102 main flow path 103, 103b sub flow path 106 second filter h hole bs cell suspension c white blood cell (tumor cell)

Claims (12)

A cell arrangement system for arranging cells in blood,
A main channel for flowing a cell suspension containing cells in the blood;
A sub-flow path having one end connected to a side surface of the main flow path;
A first filter provided between the main flow path and the sub flow path and having a filter surface having an opening smaller than the diameter of cells to be arranged;
A liquid feeding mechanism for feeding liquid in the forward direction and the reverse direction of the main flow path;
Have
By the liquid feeding mechanism, the cell suspension is fed in the forward direction of the main channel and passed over the first filter, and then the cell suspension is fed in the reverse direction of the main channel and the first channel is sent. The cell arrangement system, wherein the cells to be arranged are arranged on the first filter by passing the filter again over one filter.   The cell suspension is reciprocated a plurality of times on the first filter by repeating liquid feeding in the forward direction of the main flow path and liquid feeding in the reverse direction of the main flow path by the liquid feeding mechanism. The cell array system according to claim 1. The liquid feeding mechanism has a first liquid feeding pump for feeding in the forward direction of the main flow path and a second liquid feeding pump for feeding in the reverse direction of the main flow path,
A cell suspension is fed in the forward direction of the main flow path by the first liquid feed pump and passed over the first filter, and the passed cell suspension is passed through the main flow path by the second liquid feed pump. The cell arrangement system according to claim 1, wherein the cells to be arranged are arranged on the first filter by sending the solution in the opposite direction and passing the first filter again. The liquid feeding mechanism includes a first liquid feeding pump, and a path switching unit that switches a liquid feeding path by the first liquid feeding pump to a first path and a second path,
The cell suspension is fed in the forward direction of the main flow path by the first liquid feed pump having the liquid feed path as the first path by the path switching means, and passed over the first filter,
The passed cell suspension is fed in the direction opposite to the main flow path by the first liquid feed pump whose second path is the second path by the path switching means, and again passes over the first filter. The cell array system according to claim 1 or 2, wherein the cells to be arrayed are arrayed on the first filter. The liquid feeding mechanism has a third liquid feeding pump connected to the downstream side of the sub-flow path,
The cell arrangement system according to any one of claims 1 to 4, wherein the inside of the auxiliary flow path is set to a negative pressure by the third liquid feeding pump. The liquid feeding mechanism includes a first liquid feeding pump capable of switching a liquid feeding direction between a forward direction of the main flow path and a reverse direction of the main flow path, and a third liquid feed pump connected to the downstream side of the sub flow path. Have
In a state in which the inside of the auxiliary flow path by the third liquid feeding pump is set to a negative pressure,
A cell suspension is fed in the forward direction of the main flow path by the first liquid feed pump and passed over the first filter, and the passed cell suspension is passed through the main flow path by the first liquid feed pump. The cell arrangement system according to claim 1, wherein the cells to be arranged are arranged on the first filter by sending the solution in the opposite direction and passing the first filter again.   The main channel, which is disposed downstream of the first filter in the forward direction of the main channel, has a filter surface formed in a direction intersecting the channel axis direction, and is smaller than the diameter of the cells to be arranged The cell arrangement system according to any one of claims 1 to 6, further comprising a second filter having a small aperture. The other end different from the one end of the sub flow path is connected to the main flow path,
8. The cell suspension sent into the sub-flow path is sent from the other end to the main flow path by a third liquid feed pump. 8. Cell array system.   8. The vibration according to claim 5, wherein the first liquid is pumped by positive pressure and negative pressure by the third liquid feeding pump, and vibration is applied to the first filter by alternately repeating this. The cell arrangement system according to one item. A filter surface having a main channel, a sub channel whose one end is connected to a side surface of the main channel, and an opening smaller than the diameter of the cell to be arranged, which is provided between the main channel and the sub channel. A cell arrangement method for arranging cells on the first filter of a chip structure having a formed first filter,
A first step of passing the cell suspension over the first filter by feeding a cell suspension containing cells in blood in the forward direction of the main flow path;
And a second step of allowing the liquid to flow again in the reverse direction of the main flow path so as to pass through the first filter again.   The cell arranging method according to claim 10, wherein the first step and the second step are repeated. A pump for changing the pressure inside the sub-flow path is connected;
12. The method according to claim 10, further comprising a vibration step of applying vibration to the first filter by making the pressure inside the sub-flow path positive and negative by the pump and repeating this alternately. The cell array method described in 1.

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