JP6303496B2 - Cell deployment device - Google Patents

Description

  The present invention relates to a cell expansion device used for detection of rare cells.

  Circulating tumor cells [CTC], circulating vascular endothelial cells [CEC], circulating vascular endothelial progenitor cells [CEP], various stem cells, etc. (collectively referred to herein as “rare cells”) are contained in whole blood according to the pathological condition. It is a very rare cell. Despite the obvious clinical utility of rare cell detection, it is extremely difficult to detect. In recent years, various cell separation techniques have been applied to try and detect them.

  For example, in Patent Document 1, a cell is accommodated in each microchamber of a microchamber chip having a plurality of microchambers, and it is confirmed whether or not a specific cell exists in the accommodated cells. It is disclosed.

  In the cell deployment device 10 of Patent Document 1, as shown in FIG. 3, about 50 microchambers 4 in the vertical direction and about 200 microchambers 4 in the horizontal direction are formed in the cell deployment part A spreading on the microchamber chip 2. Is formed.

  In the cell deployment device 10 in which a plurality of microchambers 4 are formed as described above, it is disclosed that a cell suspension is added and dropped onto the microchamber chip 2. No details are given on whether to add or drop. Just guess.

  For example, in such a cell deployment device 10, as illustrated in the cross-sectional view of FIG. 4, the plate-shaped flow path forming frame body 8 that forms the flow path 5 between the flat micro chamber chip 2 is provided. The inlet portion 3 and the outlet portion 7 are formed in the flow passage forming frame 8 so as to communicate with the flow passage 5, and the cell suspension is placed in the flow passage 5 from the inlet portion 3 toward the outlet portion 7. It is conceivable to develop a cell suspension on the microchamber chip 2 by introducing.

WO2010 / 027003

However, even if the cell suspension is introduced in this manner, the flow path 5 of the cell deployment device 10 is not limited to the inlet 3 or the inlet 3 where the cell suspension is introduced, as schematically shown in FIG. The diameter of the outlet portion 7 is formed narrower than the width B of the cell deployment portion A. In such a case, since the flow rate of the liquid varies in the flow path 5, it is difficult to uniformly spread the cell suspension over the entire flow path.
The detailed reason is as follows.

  That is, if the magnitude | size of the flow rate of the device 10 for cell expansion | deployment is divided roughly, for example in FIG. 5, since the rectangular cell expansion | deployment part A has a fixed flow path width and occupies most of a flow path, The rectangular cell development part A shows an average flow velocity. This rectangular cell development portion A is defined as a first region D.

For example, in FIG. 5, in the upstream triangular portion (referred to as the second region C ₁ ) in which the channel width gradually increases from directly below the inlet portion 3 toward the rectangular cell development portion A, the cell suspension The flow rate of is higher than the average flow rate. Similarly, the downstream triangular portion (referred to as the second region C ₂ ) in which the channel width gradually narrows from the terminal portion of the cell expansion portion A toward the outlet portion 7 is also faster than the average flow velocity.

Further, the outer portion in the width direction (referred to as the third region E ₁ and the third region E ₂ ) in FIG. 5 is slower than the average flow velocity because the flow path is close to the side wall portion. Become.
As described above, in the cell deployment device 10, since the entire flow velocity in the flow path 5 varies, it is difficult to deploy the cell suspension substantially uniformly over the entire flow path.

  On the other hand, in the detection of rare cells, the cell suspension is developed on the microchamber chip 2, and then the position of the cells that have failed to enter the microchamber 4 is moved and dropped into the neighboring microchambers 4. In order to carry out intermittent liquid feeding for a period of time, so-called one-shot liquid feeding, or to bring out a part of the cells that have been stored in one microchamber 4 in layers, one-shot liquid feeding For example, the cleaning solution is continuously fed at a higher flow rate.

  However, in such a case, if the flow velocity varies depending on the position of the cell deployment device 10, the rare cells to be retained are detached without being retained, or a plurality of cells are overlapped in the microchamber 4. In other words, it is conceivable that the detectability of the cells to be detected and the number of collected cells are reduced.

  Although such a problem also occurs at the time of cell deployment, it tends to occur at the time of one-shot liquid feeding or continuous liquid feeding.

  In view of such a situation, the present invention is such that, in particular, even when a cell suspension is developed or one-shot liquid feeding or continuous liquid feeding is performed, necessary cells are detached from the microchamber. An object of the present invention is to provide a device for cell deployment that can improve the detectability and recovery rate of cells to be detected without overlapping a plurality of cells in the microchamber.

  As a result of intensive studies, the present inventors have focused on the fact that there is a difference in the flow velocity distribution generated in the device for cell deployment, and by significantly averaging the flow velocity distribution as a whole, the cell recovery rate is significantly improved. As a result, the present invention has been completed.

That is, this invention provides the device for cell expansion | deployment shown by following [1]-[ 4 ].
[1]
A microchamber chip in which a cell development region portion having a plurality of microchambers is formed;
A flow path forming frame which is provided integrally with the micro chamber and forms a flow path extending in a planar direction on the plurality of micro chambers;
An inlet part that is provided in the flow path forming frame and allows a cell suspension to flow into the flow path on the microchamber;
A cell deployment device having an outlet portion provided in the flow path forming frame and allowing a cell suspension to flow out of the flow path on the microchamber,
The shape of the microchamber is a truncated cone shape,
In a predetermined shape of the cell spreading region, a region where the cell suspension flows at an average speed is a first region, a region where the cell suspension flows faster than the first region is a second region, and the first region is the first region. When the region where the cell suspension flows slower than the region is the third region,
The depth of the micro chamber formed at the position corresponding to the second region is made deeper than the micro chamber formed at the position corresponding to the first region, and is formed at the position corresponding to the third region. A device for cell deployment in which the depth of the microchamber is shallower than that of the microchamber formed at a position corresponding to the first region.

[2]
The depth of the microchamber formed at the position corresponding to the second region is 6/5 to 10/5 the depth of the microchamber formed at the position corresponding to the first region, [1] The device for cell expansion described in 1.

[3]
The depth of the microchamber formed at the position corresponding to the third region is 1/5 to 4/5 the depth of the microchamber formed at the position corresponding to the first region. [1] Or the device for cell expansion | deployment as described in [2].

[4]
The device for cell deployment according to any one of [1] to [3], wherein an inner diameter of the microchamber is 20 to 500 μm.

  According to the present invention, the cells in the cell suspension can be appropriately captured in each microchamber. In addition, even if various liquid delivery such as one-shot liquid feeding or continuous liquid feeding is performed after spreading the cell suspension, cells may be detached from the microchamber at the local position or multiple cells may overlap. Can be prevented as much as possible.

  Thereby, it is possible to improve the detectability of cells to be examined and improve the number of cells collected.

FIG. 1 is a schematic plan view showing a microdevice according to a preferred embodiment of the present invention. FIG. 2 is a schematic cross-sectional view in the XX line direction of FIG. FIG. 3 is a schematic view schematically showing a portion through which a cell suspension flows in the cell deployment device disclosed in Patent Document 1. As shown in FIG. FIG. 4 is a schematic cross-sectional view of the cell deployment device cut along the flow path direction. FIG. 5 is a top view schematically showing a specific example of the device for cell deployment.

Hereinafter, a cell deployment device according to a preferred embodiment of the present invention will be described in detail with reference to the drawings.
In addition, this invention is applicable to the device for cell expansion | deployment provided with the flow path of various shapes. That is, the shape of the cell deployment device 10 shown as an example in FIG. 3, the shape of the flow path 5 provided in the device 10, or the path of the flow path 5 is not limited in any way. The case where the shape of the deployment device and the shape of the flow channel are similar to those in FIG. 5 will be described as an example.

  FIG. 1 is a schematic view showing a cell deployment device 30 according to a preferred embodiment of the present invention, and FIG. 2 is a sectional view thereof. In FIG. 1, the same elements as those in FIG.

  As shown in FIGS. 1 and 2, the cell deployment device 30 includes a microchamber chip 31 in which a plurality of microchambers 36 are formed, and a single flow path 35 formed on the plurality of microchambers 36. As described above, the flow path forming frame body 32 integrated with the microchamber chip 31 via the frame-shaped sealing member 34 around the periphery, the inlet portion 3 provided in the flow path forming frame body 32, and the inlet portion 3 In order to lead out the cell suspension introduced into the flow path 35 from the flow path 35, an outlet portion 7 and the like provided in the flow path forming frame 32 are provided.

  The microchamber chip 31 is also called a microchamber array [MCA]. In this embodiment, about 14,000 to 20,000 microchambers 36 are formed on the surface thereof.

A microchamber refers to a concave minute hole that can “accommodate” and “hold” one or more cells, and preferably has a bottom.
Here, “holding” means that the cells contained in the microchamber are difficult to get out of the microchamber by sending a staining solution or a washing solution to the flow path of the microchamber chip for cell deployment.

  Moreover, it is preferable that the diameter of the opening upper part of the micro chamber 36 is 20 micrometers-500 micrometers. When the diameter is in the range of 20 μm to 500 μm, the cells can be suitably accommodated and held in the microchamber 36.

  The depth of the microchamber 36 is preferably about 10 to 15 cells per microchamber, and typically the depth of the microchamber 36 is 20 μm to 500 μm.

The shape of the microchamber 36 is an inverted conical shape with a flat bottom, but is not limited thereto.
Examples of the material of the microchamber chip 31 include polymers such as polystyrene, polyethylene, polypropylene, polyamide, polycarbonate, polydimethylsiloxane [PDMS], polymethyl methacrylate [PMMA], and cyclic olefin copolymer [COC]. The microchamber chip 1 may be a combination of a plurality of materials in which a molded polymer is bonded to a substrate made of metal, glass, quartz glass, or the like.

  The method of manufacturing the microchamber chip 31 may be a method of molding using a mold having a convex portion corresponding to the shape of the microchamber 36 on the mold substrate surface, and is made of the polymer, metal, glass, or the like. A method of forming a microchamber by performing direct processing (for example, fine processing by lithography, excavation processing, LIGA process, etc.) on the substrate may be used, but from the viewpoint of productivity, a manufacturing method of forming using a mold is preferable. .

  The microchamber chip 31 can be subjected to surface treatment as necessary. Examples of the surface treatment include, but are not limited to, plasma treatment (oxygen plasma treatment or the like), corona discharge treatment, or treatment with a hydrophilic polymer, protein, lipid, or the like.

By the surface treatment, the cells are easily collected on the bottom surface without adsorbing to the inner wall of the microchamber 36, so that the cell observation in the bright field under the microscope is facilitated.
On the microchamber chip 31 of the cell deployment device 10, a flow path 35 for mainly flowing a cell suspension is formed.

The flow path 35 is not limited to this example as long as the cell suspension can be circulated on the microchamber chip 31.
In the example of the flow path 35 shown in FIGS. 1 and 2, the flow path is formed by a flow path forming frame 32 and a seal member 34 that are provided integrally with the micro chamber chip 31 with the upper surface of the micro chamber chip 31 as the bottom surface. A path 35 is defined.

  The flow path 35 communicates with the inlet portion 3 and the outlet portion 7 of the flow path forming frame 32, and the cell suspension that has flowed in from the inlet portion 3 flows in the direction indicated by the arrow, from the outlet portion 7. It can be derived.

  From the viewpoint of ease of observation and maintenance, the microchamber chip 31 and the flow path forming frame body 32 are preferably configured to be attachable to and detachable from each other by means such as engagement, screw fixing, and adhesion.

  The height T of the flow path 35, that is, the distance from the surface portion other than the microchamber 36 of the microchamber chip 31 to the ceiling (hereinafter also referred to as “ceiling height”) is preferably 50 μm to 500 μm. .

  When the height of the ceiling is in the range of 50 μm to 500 μm, the cells attached to the surface portion other than the microchamber 36 of the microchamber chip 31 can be easily moved by the force of the liquid flow. In addition, the time until the cells settle on the surface of the microchamber chip 31 can be shortened. Further, it is preferable because clogging of cells in the channel is less likely to occur, and the cells can be smoothly developed.

As the material of the flow path forming frame 32, for example, the same material as that of the microchamber chip 31 is preferably used.
Here, the flow path forming frame 32 is suitably combined with the same surface treatment as the above-described microchamber chip 31 (electric treatment of plasma treatment or corona discharge treatment, or impurities other than the target rare cells). Such a coating treatment with a hydrophilic polymer, protein, lipid or the like may be performed.

  The cell suspension may contain rare cells, for example, blood such as human, lymph fluid, tissue fluid, body cavity fluid, etc., and may be appropriately diluted with a diluent or the like. The cell suspension is not limited to those derived from living organisms, and may be a cell dispersion prepared by suspending cells artificially for testing and research. In particular, application of a cell suspension after separating red blood cells from blood is suitable for collecting and detecting rare cells such as CTC.

  Examples of rare cells include cancer cells. In particular, when the cell suspension is blood or a blood-derived specimen, the rare cells are any of CTC (circulating tumor cells or circulating cancer cells), CEC (circulating vascular endothelial cells) and CEP (circulating vascular endothelial progenitor cells). One or more types of cells may be used.

The diameter of various cells contained in such a cell suspension is preferably 10 to 100 μm, but it is necessary to be at least smaller than the diameter of the microchamber 36.
Each element such as the microchamber chip 31, the flow channel forming frame 32, the flow channel 35, and the cell suspension of the cell deployment device 10 in the present embodiment is configured as described above. The configuration of the main part will be further described.

  Now, in the cell deployment device 30, it is assumed that the depths of the microchambers 36 are all the same. In such a case, the flow rate of the cell suspension introduced from the inlet portion 3 and led out at the outlet portion 7 is such that the flow rate of the cell suspension is the first located in the substantially central portion of the microchamber chip 31 as described above. One region D shows an average flow velocity V.

In the second region C ₁ following the first region D from the inlet 3, it becomes faster than the average flow velocity V. In addition, the second region C ₂ from the end portion of the first region D toward the outlet portion 7 is also faster than the average flow velocity V.

On the other hand, since the side wall portion of the seal member 34 is located in both width direction outer side portions than the first region D, that is, in the third region E ₁ and the third region E ₂ , it is slower than the average flow velocity V. Become.
Therefore, the present inventor sets, for example, a region indicating the average flow velocity V as the region D as the first region, and sets a region faster than the average flow velocity V as the regions C ₁ and C ₂ as the second region, When a region slower than the average flow velocity V such as regions E ₁ and E ₂ is defined as the third region, the depth of the plurality of microchambers included in the second region is increased, and the third region is further increased. It has been found that if the depth of the plurality of contained microchambers is set to be shallow, the entire flow velocity of the flow path 35 is averaged.

Therefore, according to the present embodiment, the depths of the microchambers 36 included in the first region D showing the average flow velocity V are all the same depth, and in the second region C ₁ and the second region C ₂ , The depths of the plurality of micro chambers 36 included in the regions C ₁ and C ₂ are set to be deeper than the depth of the micro chambers 36 in the first region D, and the third region E ₁ and the third region The depth of the plurality of microchambers 36 included in E ₂ was set to be shallower than the depth of the microchambers 36 included in the first region D.

As a specific ratio, the depth of the microchamber 36 included in the second region C ₁ and the second region C ₂ is 6/5 compared with the depth of the microchamber 36 formed in the first region D. It is preferable to set deeply at a ratio of -10/5.

Further, the depth of the microchamber 36 included in the third region E ₁ and the third region E ₂ is a ratio of 1/5 to 4/5 as compared with the depth of the microchamber 36 included in the first region D. It is preferable to set shallowly.

Thus, if the depth of the micro chamber 36 in each region is set, it becomes possible to appropriately capture cells in each micro chamber 36.
With such a setting, even if various liquid feedings were performed after cell expansion, it was prevented that cells were detached from each microchamber 36 or a plurality of cells overlapped. In addition, according to the experiment, it was examined how much the portion where the cells were normally stored was, and in this embodiment, the detection sensitivity was improved by 15 to 20% in the area ratio.

As mentioned above, although preferable embodiment of this invention was described, this invention is not limited to this embodiment at all.
For example, in the above embodiment, the inlet portion 3 arranged on one side of the microchamber chip 31 and the outlet portion 7 arranged on the other side of the microchamber chip 31 are configured in a linear arrangement. Instead of this, for example, the inlet portion 3 is arranged at the center of the microchamber chip 31, and the solution introduced from the inlet portion 3 spreads radially, for example, and the solution is supplied from one or a plurality of outlet portions. The present invention is also applicable to a cell deployment device having a flow path from which is derived.

In general, the present invention is generally set so that the depth of the microchamber 36 in the portion where the flow velocity is high is deepened and the depth of the microchamber 36 in the portion where the flow velocity is low is shallow. The shape of the flow path as viewed in plan is not limited at all.

  Hereinafter, although an example about the present invention is given and explained in detail, the present invention is not limited to this example.

(1) Preparation of cell suspension 2 × 10 ⁵ human leukemia cells (CEM) were added to phosphate buffer (PBS), and 10 ³ human breast cancer cells (MDA-MB-231) were further added. The final volume was adjusted to 68 μl.

(2) Creation of a cell deployment device Acrylic (including an inlet and an outlet) so as to form a flow path having a length of 15 mm, a width of 68 mm, and a height of 100 μm on the surface of the microchamber chip on which the microchamber is formed. A flow path forming member made of PMMA) and a microchamber chip were bonded together with a seal member (double-sided seal) having a thickness of 100 μm to form a device for cell deployment as shown in FIG.

(3) Preparation of cell staining solution 1 μg / ml of Alexa647-labeled anti-human cytokeratin antibody was added to a PBS solution containing 3% BSA and 1% Tween to obtain a cell staining solution.

(4) Cell detection step Using the above cell expansion device, 150 μl of 30% ethanol solution is injected from the inlet side at a flow rate of 1 ml / min, and then 450 μl of PBS solution is injected at 1 ml / min. Air bubbles were removed. Next, 68 μm of the prepared cell suspension was injected at a flow rate of 1 ml / min from the inlet side, and the cells were introduced into the cell expansion device. After allowing to stand for 1 minute to allow the cells to self-weight, intermittent liquid feeding was continuously performed 10 times, and the cells were introduced into the microchamber. Next, 150 μl of the cell staining solution was fed at a flow rate of 100 μl / min, allowed to stand for 30 minutes for staining reaction, and 450 μl of PBS was fed at 100 μl / min to remove the staining reagent. Finally, the number of cells in the microchamber excited at a wavelength of 650 nm was counted using a fluorescence microscope to obtain the number of detected cells.

  The reagent is injected from the inlet portion 3 into the device for cell deployment, and after passing through the bubble removal process in the microchamber, the cell introduction process, the process of storing the cells in the microchamber, and the process of staining the cells, The number of MDA-MB231 cells stored therein was calculated. In addition, when cells in the microchamber within 1 mm from the channel wall were observed, cell overlap was observed, and cell detachment was observed when the cells in the microchamber at the channel enlarged portion and the channel reduced portion were observed. It was.

[Example]
In the inlet portion (second region C ₁ ) where the channel width gradually increases and the outlet portion (second region C ₂ ) where the channel width gradually decreases, the opening is 120 μm, the bottom surface is 90 μm, and the depth is 100 μm. In the central part (first region D ₁ ) where the chamber is arranged and the flow path width is uniform, a microchamber having an opening of 120 μm, a bottom of 90 μm and a depth of 50 μm is arranged, the opening is 120 μm, and the bottom A microchamber having a diameter of 90 μm and a depth of 10 μm was arranged in a range from the flow path wall surface to 1 mm (third region E ₁ , E ₂ ), and a flow path height of 100 μm was used.

  The reagent was injected from the inlet part side, passed through the bubble removal process in the microchamber, the cell introduction process, the process of storing the cells in the microchamber, and the process of staining the cells, and stored in the microchamber with a fluorescence microscope. When the number of cells was calculated and compared with the number of cells in the comparative example, the number of cells increased by 15%.

[Comparative example]
A microchamber having an opening of 120 μm, a bottom surface of 90 μm, and a depth of 50 μm was disposed in the entire flow path, and a microdevice having a flow path height of 100 μm was used.
Note that the depths of the microchambers in the comparative example were all 90 μm.

3 Inlet part 7 Outlet part 30 Cell development device 32 Channel forming member 35 Channel 36 Microchamber A Cell development part C ₁ , C ₂ Second region D First region E ₁ , E ₂ Third region T height

Claims (4)

A microchamber chip in which a cell development region portion having a plurality of microchambers is formed;
A flow path forming frame which is provided integrally with the micro chamber and forms a flow path extending in a planar direction on the plurality of micro chambers;
An inlet part that is provided in the flow path forming frame and allows a cell suspension to flow into the flow path on the microchamber;
A cell deployment device having an outlet portion provided in the flow path forming frame and allowing a cell suspension to flow out of the flow path on the microchamber,
The shape of the microchamber is a truncated cone shape,
In a predetermined shape of the cell spreading region, a region where the cell suspension flows at an average speed is a first region, a region where the cell suspension flows faster than the first region is a second region, and the first region is the first region. When the region where the cell suspension flows slower than the region is the third region,
The depth of the micro chamber formed at the position corresponding to the second region is made deeper than the micro chamber formed at the position corresponding to the first region, and is formed at the position corresponding to the third region. A device for cell deployment in which the depth of the microchamber is shallower than that of the microchamber formed at a position corresponding to the first region.   The depth of the micro chamber formed at a position corresponding to the second region is 6/5 to 10/5 of the micro chamber formed at a position corresponding to the first region. The device for cell expansion described in 1.   The depth of the microchamber formed at the position corresponding to the third region is 1/5 to 4/5 the depth of the microchamber formed at the position corresponding to the first region. Or the device for cell expansion | deployment of 2 or 2.   The device for cell deployment according to any one of claims 1 to 3, wherein an inner diameter of the microchamber is 20 to 500 µm.

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