JP2018061447A - Method of recovering rare cells - Google Patents

Abstract

PROBLEM TO BE SOLVED: To allow the recovery of rare cells from a rare-cell-containing liquid at high yield.SOLUTION: The rare cell recovery method uses a cell capture device provided with a housing having an inflow port and an outflow port, and a filter with a plurality of through holes on a flow path within the housing between the inflow port and the outflow port, and comprises: a capturing step S01 of capturing rare cells by filtering the rare-cell-containing liquid using a filter by introducing the rare-cell-containing liquid through the inflow port and discharging from the outflow port; a buffer replacement step S03 of filling the flow path in the cell capture device with buffer; a resin replacement step S04 of replacing the buffer in the flow path in the cell capture device with a photo-curable resin; a transfer step S05 of curing the photo-curable resin in the cell capture device thereby transferring the rare cells from the filter to the photo-curable resin; and a recovery step S06 of removing the photo-curable resin cured in the transfer step from the cell capture device and recovering the cells.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a method for collecting rare cells.

  Cancer accounts for the top cause of death in countries around the world, and more than 300,000 people die from cancer annually in Japan. Most cancer deaths are due to recurrence of cancer metastasis. Cancer metastasis recurrence is that cancer cells settle from the primary lesion via the blood vessels or lymph vessels to the blood vessel wall of another organ tissue, infiltrate the blood vessel wall, and form a fine metastasis layer on the blood vessel wall. Occur. Thus, cancer cells circulating in the body via blood vessels or lymphatic vessels are called circulating tumor cells (CTC).

  On the other hand, circulating endothelial cells (CEC) are present in peripheral blood. CEC is a mature cell in which endothelial cells are detached from the blood vessel wall due to metabolism, and it is known that the number of CEC increases due to many diseases such as cardiovascular disease, infectious disease, immune disease and cancer.

  As described above, since a lot of information on diseases can be obtained from rare cells such as CTC and CEC, the presence of these rare cells and early detection of the increase are expected.

However, blood contains many blood cell components such as red blood cells, white blood cells, and platelets, and the number is said to be 3.5 × 10 ^{9 to} 9 × 10 ^{9 in} 1 mL of blood. On the other hand, since there are only a few rare cells in 1 mL of blood, detection and observation of rare cells is very difficult. In order to efficiently detect rare cells in the presence of many blood cell components, it is necessary to separate the rare cells from the blood cell components.

  As a method for separating rare cells in blood, there is a method using a filter (for example, Patent Documents 1 to 3). This method is a method for separating rare cells based on differences in cell size and deformability.

JP 2013-42689 A JP 2011-163830 A International Publication No. 2015/012315

  As a recent trend, there is an increasing expectation that the results of genetic analysis of isolated rare cells will be used for, for example, selection of optimal drugs and prognosis. In PCR and next-generation sequencer (NGS) used for gene analysis, the amount and purity of rare cells that are the basis of analysis are important.

  However, even if the conventional methods such as Patent Documents 1 to 3 are used, it is difficult to recover the rare cells captured on the filter, and the rare cells cannot be isolated with sufficient yield.

  This invention is made | formed in view of said subject, and it aims at providing the rare cell collection | recovery method which can collect | recover rare cells with a high yield from a rare cell containing liquid.

  In order to achieve the above object, the rare cell recovery method according to the present invention includes a housing having an inlet for introducing a liquid into the interior and an outlet for discharging the liquid to the outside, and the inlet. A rare cell recovery method using a cell trapping device in which a filter having a plurality of through holes in a thickness direction is provided on a flow path inside the housing between the outlet and the outlet. The rare cell-containing liquid is introduced into the rare cell-containing liquid on one surface of the filter by introducing the rare cell-containing liquid from the outlet and discharging the rare cell-containing liquid through the outlet and filtering the rare cell-containing liquid with the filter. A trapping step for trapping, a buffer replacement step for filling the flow path in the cell trapping device with a buffer by introducing a buffer from the inlet, and a photocurable resin being introduced from the inlet A resin replacement step of replacing the buffer of the flow path in the cell trapping device with the photocurable resin; and curing the photocurable resin in the cell trapping device to remove the rare cells into one of the filters. A transfer step of transferring the photocurable resin from the surface to the photocurable resin, and a recovery step of taking out the photocurable resin cured in the transfer step from the cell trapping device.

  According to the above rare cell recovery method, after introducing rare cell-containing liquid into the cell capture device to capture the rare cells with a filter, the photocurable resin is introduced into the cell capture device and cured. Thus, the rare cells can be transferred to the photocurable resin and recovered. In this way, by introducing a photocurable resin around the rare cells and curing them, the rare cells can be recovered while suppressing the migration of the rare cells, thus preventing the rare cells from being lost. Cells can be collected. Furthermore, in the above rare cell recovery method, the photocurable resin is introduced into the cell trapping device by replacing the buffer and the photocurable resin after filling the flow path in the cell trapping device with the buffer. . By adopting such a procedure, when the photocurable resin is introduced, the rare cells are prevented from moving from the surface of the filter into the through-hole, so that the yield of the rare cells can be further increased. .

  Here, the said photocurable resin can be set as the aspect whose transmittance | permeability of visible light after hardening is 20% or more.

  As described above, when a photocurable resin having a visible light transmittance of 20% or more after curing is used, the rare cells are observed while the rare cells are retained in the photocurable resin collected in the collecting step. It becomes possible to do.

  Moreover, in the said collection | recovery process, it can be set as the aspect which collects the said photocurable resin equivalent to the one surface side of the said filter separately from the said other photocurable resin.

  As described above, the photocurable resin corresponding to one surface side of the filter is collected separately from the other photocurable resins, so that the rare cells can be observed or removed from the photocurable resin. It is possible to suitably perform subsequent operations such as analysis of rare cells.

  The recovery step may further include a step of individually recovering the rare cells from the photocurable resin taken out from the cell capture device.

  As described above, by including the step of individually collecting rare cells, it becomes possible to perform more detailed analysis relating to rare cells such as gene analysis.

  ADVANTAGE OF THE INVENTION According to this invention, the rare cell collection | recovery method which can collect | recover rare cells with a high yield from a rare cell containing liquid is provided.

It is a flowchart explaining a series of operation procedures including the rare cell collection | recovery method concerning one Embodiment of this invention. It is a schematic diagram which shows the structure of a cell capture device. It is a schematic diagram which shows a resin substitution process, a transfer process, and a collection | recovery process.

  DESCRIPTION OF EMBODIMENTS Hereinafter, embodiments for carrying out the present invention will be described in detail with reference to the accompanying drawings. In the description of the drawings, the same elements are denoted by the same reference numerals, and redundant description is omitted.

  The rare cell recovery method according to an embodiment of the present invention is a method in which a rare cell-containing solution is filtered by a filter held in a cell trapping device, so that the rare cell is captured by the filter, and then the rare cell is photocurable. It is characterized by being transferred to a resin and collected. Since the cell capture device is used to transfer and recover the photocurable resin, it is possible to collect rare cells while preventing loss of rare cells captured on the filter. Recovery with high yield is possible.

  The “rare cell” to be collected in the rare cell collection method according to the present embodiment is a specific type of cell contained in a biological sample, and the ratio of the number of cells is usually in the rare cell-containing solution. It is extremely small relative to the total number of all cells contained. The rare cells can be, for example, cancer cells such as CTC (Circulating Tumor Cell) and endothelial cells such as CEC (Circulating Endothelial Cell), but are not limited thereto.

  The rare cell-containing liquid is a liquid containing the above rare cells, and can be, for example, blood, ascites, pleural effusion, and other cell suspensions. The cell suspension can be, for example, a liquid containing leukocytes. The rare cell-containing solution may further contain a buffer or an additive.

  “Capture” in the present embodiment means that the rare cell-containing solution is filtered through a filter, and the rare cells remain on the filter.

  In addition, “transfer” in the present embodiment refers to a transition from a state where rare cells are captured by a filter to a state where rare cells are retained by a photocurable resin. That is, by transferring the rare cells from the filter to the photocurable resin, the rare cells are held in the photocurable resin.

  Further, “recovery” in the present embodiment refers to recovering rare cells. However, the invention is not limited to collecting rare cells alone, and collecting photocurable resin in a state in which rare cells are retained also falls under “recovery” in the present embodiment.

  Hereinafter, the rare cell collection method according to the present embodiment will be described with reference to FIGS. As shown in FIG. 1, the rare cell recovery method according to the present embodiment includes a capture step (step S01), a washing step (step S02), a replacement step (buffer replacement step: step S04), and a resin replacement step (step S04). , A transfer step (step S05), a recovery step (step S06), and an analysis step (step S07).

The capturing step S01 is a step of capturing rare cells on one surface of the filter by filtering the rare cell-containing liquid through a filter attached to the cell capturing device. The cleaning step S02 is a step of cleaning the filter surface with a cleaning liquid (wash buffer).
The replacement step S03 is a step of replacing the area around the filter in the cell trapping device with a buffer such as a washing solution. In addition, the resin replacement step S04 is a step of introducing a pre-curing photocurable resin into the region around the filter in the cell trapping device. The transfer step S05 is a step of transferring the rare cells on the surface of the filter to a state where the photocurable resin is holding by curing the photocurable resin on the filter surface. The recovery step S06 is a step of recovering the photocurable resin in which the rare cells are retained, and recovering the rare cells from the photocurable resin as necessary. The analysis step S07 is a step of performing an analysis related to the collected rare cells. Details of each step will be described later.

  First, the capturing step S01 will be described. The capturing step S01 is a step of capturing rare cells on the surface of the filter by filtering the rare cell-containing solution through a filter. The rare cell-containing liquid is a liquid containing rare cells to be captured, and includes, for example, blood or blood-derived liquid, but is not limited thereto.

  When performing filtration, a cell capture device is used. The cell trapping device will be described with reference to FIG.

  FIG. 2 is a schematic diagram showing the configuration of the cell trapping device. The cell trapping device 100 includes a housing 120 having an inflow port 130 to which an inflow pipe 125 into which liquid flows in is connected, an outflow port 140 to which an outflow pipe 135 through which liquid flows out is connected, and a filter 105. The shape of the housing 120 may be a rectangular parallelepiped or a cylinder, and is not particularly limited. The filter 105 is fixed by a casing 120 composed of an upper member 110 and a lower member 115, and is disposed on a flow path inside the casing 120 formed between the inlet 130 and the outlet 140. .

  The upper member 110 and the lower member 115 are fixed to each other by, for example, a clip (not shown), and the filter 105 can be taken out by removing the clip and disassembling the housing 120. In the rare cell collection method according to the present embodiment, the photocurable resin is introduced into the space sandwiched between the upper member 110 and the lower member 115, so that the cured photocurable resin can be collected. It is preferable that As shown in FIG. 2, the space between the upper member 110 and the lower member 115 is formed by the upper space A1 formed by the upper member 110 and the filter 105, and the lower member 115 and the filter 105. This is the lower space A2.

  A reservoir containing a treatment liquid such as a rare cell-containing liquid or a washing liquid is connected to the upstream of the inflow pipe 125. The liquid that has entered the reservoir flows from the inflow pipe 125 into the housing 120, passes through the filter 105, and is discharged from the outflow pipe 135 to the outside. Such a liquid flow can be created by driving a pump P connected downstream of the outflow pipe 135. Further, the movement speed (flow velocity) of the liquid in the cell trapping device 100 can be controlled by driving the pump P. As the pump P, for example, a peristaltic pump or the like can be used.

  The filter 105 is formed with a plurality of through holes 106 penetrating in the thickness direction. For example, when blood containing cancer cells is introduced into the cell capture device 100 as a rare cell-containing solution, components such as blood platelets and red blood cells pass through the through-hole 106, but rare cells and some white blood cells pass through the through-hole 106. Cannot pass through, and remains on one surface (the surface on the inflow pipe 125 side) on the filter 105.

  The filter 105 is not particularly limited as long as it has a structure in which a number of through holes are formed in a thin film of plastic or metal, and a conventionally known filter can be used. The filter 105 removes components other than the rare cells from the rare cell-containing solution together with the filtrate, and captures the rare cells that have not passed through the through-hole 106 on the surface thereof. For example, when using blood containing cancer cells as the rare cell-containing solution, by using a filter having a substantially rectangular through-hole 106 having a size of about 8 μm (short side) × 100 μm (long side), Blood components such as red blood cells, white blood cells, and platelets can be removed as filtrate, and cancer cells that are rare cells can be captured by the filter 105. In addition, the hole diameter of the through-hole 106 provided in the filter 105 can be appropriately changed according to the type of cells to be captured. Further, the shape of the through hole 106 can be appropriately changed. Examples of the shape of the through hole 106 include an ellipse, a square, a rectangle, a rounded rectangle, and a polygon. From the viewpoint of cell capture efficiency, a circle, rectangle, or rounded rectangle is preferable. A rounded rectangle is a shape composed of two long sides of equal length and two semicircles.

  The flow rate when the liquid such as the rare cell-containing liquid and the washing liquid passes through the inflow tube 125 is preferably 100 μL / min to 800 μL / min, and more preferably 200 μL / min to 600 μL / min. When the flow rate of the liquid is within the above range, damage to rare cells can be further reduced, and the recovery rate of rare cells can be improved.

  The specific configuration of the cell trapping device 100 is not particularly limited as long as the rare cell-containing liquid can pass through the through-hole 106 of the filter 105.

  Next, the cleaning process (step S02) will be described. The cleaning step S02 is a step of cleaning the filter surface with a cleaning liquid. The cleaning step S02 is a step performed as necessary, and may be omitted.

  First, a washing solution is introduced from the inflow pipe 125 of the cell trapping device 100, and the filter 105 that has captured rare cells is washed with the washing solution. The washing liquid is not particularly limited as long as it can wash away components other than the rare cells remaining on the filter 105. Examples of the cleaning liquid include a phosphate buffer. The amount of the washing solution is not particularly limited, but can be about twice the amount of the rare cell-containing solution.

  When the filter 105 is washed with the cleaning liquid, the flow rate of the cleaning liquid is not particularly limited, but it is preferable to set the flow rate so that the rare cells can be prevented from moving into the through-hole 106 as the cleaning liquid moves. That is, the flow rate of the cleaning liquid introduced from the inflow pipe 125 is preferably 100 μL / min to 800 μL / min, and more preferably 200 μL / min to 600 μL / min.

  Note that it is preferable to maintain a state in which the cleaning liquid is not removed from the surface of the filter 105 after the filter 105 is cleaned with the cleaning liquid. By maintaining the state in which the cleaning liquid remains on the surface of the filter 105 and maintaining the state in which the rare cells on the surface of the filter 105 are not exposed, drying of the rare cells can be prevented. Further, it is possible to prevent the rare cells from moving into the through-hole 106 as the cleaning liquid moves. In order to create a state where the cleaning liquid is not removed from the surface of the filter 105, the flow of the cleaning liquid may be stopped by controlling the flow rate of the cleaning liquid by driving the pump P of the cell trapping device 100.

  The replacement process (step S03), the resin replacement process (step S04), and the transfer process (step S05) will be described with reference to FIG.

  The replacement step (step S03) is a step of filling the upper space A1, the lower space A2, and the through hole 106 in the cell trapping device 100 with a buffer. FIG. 3A shows a state in which rare cells C are captured on the surface of the filter 105. The rare cell C is captured by the filter 105 in a state of being present on the through hole 106 of the filter 105 or on the surface of the filter 105. In this state, the buffer is introduced into the cell capturing device 100 so that the space in the cell capturing device 100 is filled with the buffer. As the buffer, a buffer solution that does not affect the rare cells C such as a phosphate buffer solution is used, but a washing solution (wash buffer) may be used as the buffer. Moreover, you may use water as a buffer.

  The flow rate of the buffer when supplying the buffer to the cell capture device 100 is not particularly limited, but it is preferable to set the flow rate so that the rare cells can be prevented from moving into the through-hole 106 as the buffer moves. That is, the flow rate of the buffer introduced from the inflow pipe 125 is preferably 100 μL / min to 800 μL / min, and more preferably 200 μL / min to 600 μL / min.

  In FIG. 3A, the upper member 110 and the lower member 115 are omitted, and the upper space A1 and the lower space A2 are shown. The region formed by the upper space A1, the lower space A2, and the filter 105 is a region corresponding to a flow path formed between the inlet 130 and the outlet 140 in the cell trapping device 100. 3A shows a state in which the upper space A1, the lower space A2, and the through-hole 106 of the filter 105 are filled with the buffer B through the replacement step S03. . In this manner, by replacing the inside of the cell trapping device 100 with the buffer B, bubbles remaining in the cell trapping device 100 can be reduced.

  The resin replacement step S04 is a step of introducing a photocurable resin having fluidity into the flow path in the cell trapping device 100 and replacing the internal buffer. In this embodiment, the cell trapping device 100 is filled with the buffer B before introducing the photocurable resin. Therefore, the resin replacement step S03 is a step of filling the cell capture device 100 with the photocurable resin R by replacing the buffer B in the cell capture device 100 with the photocurable resin R. FIG. 3B shows a state where a photocurable resin is introduced into the cell trapping device 100. As shown in FIG. 3B, the upper space A1, the lower space A2, and the through hole 106 in the cell trapping device 100 are filled with the photocurable resin R. As a result, the periphery of the rare cell C existing on the surface of the filter 105 is covered with the photocurable resin R. Note that it is preferable to replace the buffer 105 with the photocurable resin R without exposing the filter 105 and its surroundings to air.

  The photocurable resin in the present embodiment refers to a resin that changes from liquid to solid by the action of light energy. Examples of the photocurable resin include, but are not limited to, an ultraviolet curable resin that changes to a solid upon irradiation with ultraviolet rays. As the photocurable resin, for example, an acrylic resin having biocompatible polyethylene glycol can be used. Specific examples include polyethylene glycol dimethacrylate, polyethylene glycol acrylate, ethoxylated ethylhexyl polyethylene glycol, butoxylated polyethylene glycol, and methoxypolypropylene glycol methacrylate. Moreover, it is preferable that these photocurable resins contain a photopolymerizable initiator.

  In addition, as for the photocurable resin which concerns on this embodiment, it is preferable that the transmittance | permeability of visible light (wavelength range 380 nm-780 nm light) is 20% or more about resin after hardening. When the visible light transmittance in the cured resin is 20% or more, the visibility of the rare cells C is improved even when the resin is held in the photocurable resin. Therefore, it is possible to observe the rare cells C using visible light while being held in the photocurable resin. In addition, when the transmittance of light of a specific wavelength included in the wavelength range of 380 nm to 780 nm is 20% or more, the light of the wavelength is used because it has high transparency to the light of the wavelength. It is suitably used for observation and the like.

  The flow rate of the photocurable resin R when supplying the photocurable resin R to the cell trapping device 100 is not particularly limited, but rare cells move into the through-hole 106 as the photocurable resin R moves. It is preferable to set the flow rate so that this can be suppressed. That is, the flow rate of the photocurable resin R is preferably 100 μL / min to 800 μL / min, more preferably 200 μL / min to 600 μL / min, and still more preferably 40 μL / min to 80 μL / min. .

The transfer step S05 is a step of curing the photocurable resin introduced into the cell trapping device 100. By irradiating the photocurable resin R with predetermined light, the photocurable resin R is cured. As a result, a part of the rare cells C is held by the cured photocurable resin R. That is, the photocurable resin R and the rare cells C are integrated. The amount of light applied to the photocurable resin R is appropriately set according to the conditions under which the photocurable resin R can be cured. In addition, since excessively irradiating light with respect to the photocurable resin R leads to irradiating the same amount of light to the rare cells C, there is a possibility of some influence on the rare cells C. is there. Therefore, it is preferable to set the light curable resin R within a curable range and a small amount of light. Specifically, it is preferable that the light irradiating the photocurable resin R light quantity (integrated quantity of light) is 10~1000mJ / cm ^2, more preferably 50 to 500 mJ / cm ^2, 100 to More preferably, it is 300 mJ / cm ² .

  The recovery step S06 is a step of recovering the photocurable resin R. In the present embodiment, since the photocurable resin R is cured in the cell trapping device 100, it is necessary to take out the photocurable resin R from the cell trapping device 100. Further, as shown in FIG. 3B, since the photocurable resin R is cured in a state where the upper space A1, the lower space A2, and the through hole 106 are filled, the photocurable resin R is integrated with the filter 105. It hardens in the state of becoming. Therefore, the cell trapping device 100 is disassembled to take out the hardened photocurable resin R, and portions of the photocurable resin R corresponding to the upper space A1 are taken out as needed.

  FIG. 3C shows a state in which a portion of the photocurable resin R corresponding to the upper space A1 is taken out. As shown in FIG. 3C, the rare cells C are detached from the filter 105 and moved to the side of the photocurable resin R corresponding to the upper space A1 (transfer). Note that the method for separating the portion of the photocurable resin R corresponding to the upper space A1 from the cell capturing device 100 and the filter 105 is not particularly limited. For example, when the housing 120 is separated into the upper member 110 and the lower member 115 after the photocurable resin R is cured, the photocurable resin R and the filter 105 are separated from the lower member 115 in a state where they are attached to the upper member 110 side. The At this time, since the photocurable resin R and the filter 105 are adsorbed by surface tension, the photocurable resin R and the filter 105 can be easily separated. When the filter is removed, the cells are exposed from the photocurable resin R, making it easy to remove. Moreover, in the case of said process, you may perform the below-mentioned collection | recovery process in the state in which the photocurable resin R adhered to the upper member 110. FIG. When the boundary between the photocurable resin R and the filter 105 is in close contact, the photocurable resin corresponding to the upper space A1 is removed after removing the upper member 110 and the lower member 115 of the cell trapping device 100. By inserting a jig or the like at the end of the boundary between R and the filter 105, the two can be separated. Further, after removing the upper member 110 and the lower member 115 of the cell trapping device 100, a tape or the like is applied to the upper surface of the photocurable resin R corresponding to the upper space A1 (the surface opposite to the surface on the filter 105 side). And a method of separating the photocurable resin R from the filter 105 by using this tape. In addition, the photocurable resin R separated from the filter 105 may move onto, for example, a slide glass. In this case, workability in a recovery process described later is improved.

  Furthermore, in the collecting step S06, the rare cells C may be taken out from the photocurable resin R and collected individually as necessary. Specifically, the rare cells C can be individually collected by moving the rare cells C held in the photocurable resin R to another container or the like while using a microscope as necessary. The method for collecting rare cells C is not particularly limited, but a method using a microdissector or a micromanipulator is preferred. By using a microdissector or a micromanipulator, the rare cells C can be individually collected one by one, and therefore, the gene analysis and the like can be performed on the individually collected rare cells C. Microdissector is a technique for isolating cells by flying them up. A micromanipulator is a technique for pinching cells with something like fine tweezers. As the microdissector and the micromanipulator 300 used for isolation of rare cells, known ones can be used.

  Then, an analysis process (step S07) is implemented as needed. The analysis step S07 is a step of performing an analysis related to the rare cell C isolated by a series of steps from the capture step S01 to the recovery step S06.

  In the collecting step S06, when the rare cell C held in the photocurable resin R is collected, in the analysis step, for example, the rare cell C may be observed while being held in the photocurable resin R. it can. In particular, when the visible light transmittance in the photocurable resin R is 20% or more, even when the rare cells C are held in the photocurable resin R, the photocurable resin R is preferably observed. It can be carried out.

  Genetic analysis can also be performed as a method for analyzing isolated rare cells C. In this case, the gene information of the rare cell C is obtained from the isolated rare cell C by using a known gene analysis method such as NGS (Next Generation Sequencer). The obtained genetic information can be used for various studies, tests or diagnoses. Further, the rare cell C can be classified by genotype based on the obtained genetic information. It is also possible to culture cancer cells classified by genotype and provide them as cancer cell samples of a predetermined concentration. Thus, the present invention can be widely applied in the fields of medicine and biology.

  In addition, the rare cell collection | recovery method of this embodiment can further include the process of dye | staining a rare cell other than the above process from a viewpoint which distinguishes a rare cell and another cell. The step of staining the rare cell C is not particularly limited as long as it is before the rare cell C is isolated and recovered from the photocurable resin R. For example, before the capture step S01, before the washing step S02, the replacement step It can be performed before S03, before the resin replacement step S04, or the like. The method for staining rare cells C is not particularly limited, and for example, fluorescent staining and Giemsa staining can be used.

  According to the method for recovering rare cells C of the present embodiment, the cells are flattened so that the rare cells C can be easily transferred from the rare cell-containing solution by filtration with the filter 105 in the cell trapping device 100 with good yield. Can be captured in a distributed state. Then, by introducing the photocurable resin R to the surface of the filter 105 and curing it, the rare cells C are transferred to the photocurable resin R, thereby affecting the rare cells C and the loss of the rare cells C. Since the rare cells C can be recovered while minimizing them, the rare cells can be recovered with a high yield. Moreover, the movement of the rare cell C can be regulated by introducing the photocurable resin R around the rare cell C and curing it. Therefore, loss of rare cells can be suppressed and the yield can be increased.

  Furthermore, in the method for collecting rare cells C according to the present embodiment, the cell trapping device 100 including the surface of the filter 105 is replaced by replacing the buffer and the photocurable resin after replacing the inside of the cell trapping device 100 with a buffer. A photocurable resin R is introduced into the inside. In this way, the cell trapping device 100 is filled with the buffer and then replaced with the photocurable resin R, so that when the photocurable resin R is introduced, the rare cells C are introduced from the surface of the filter 105 through the through-holes. Migration into 106 is prevented. Therefore, the yield of rare cells C can be further increased. The reason why the rare cells C are less likely to enter the through-hole 106 by replacing the buffer with the photo-curable resin R is, for example, that the replacement from the buffer causes the filter 105 to move in the thickness direction of the filter 105 due to the surface tension of the buffer. It can be considered that force can be prevented from being applied. In addition, rare cells C are likely to be deformed into a flat shape in the gas phase (dry state), whereas in a water environment (wet environment), they tend to maintain a spherical shape and are replaced from a buffer. Thus, it can be considered that the rare cell C can be prevented from touching the air (gas phase) and deteriorating the cell membrane.

  Further, conventionally, as a method for collecting rare cells after being captured by a filter, a method for solidifying and collecting a liquid surrounding the cells on the filter, such as an agarose gel method or a paraffin fixing method, has been used. The method using a photocurable resin has various advantages compared with the above method. For example, the agarose gel method in which the entire liquid is coagulated using low melting point agarose is a simple method with a low risk of cell loss. However, since the melting point of agarose is high, handling is difficult and the recovery rate is low. Moreover, the paraffin fixation method has high reliability by using paraffin that has been used for a long time, but since the melting point is high, there is a concern about the influence on cells. In contrast, in the method for collecting rare cells C according to the present embodiment, transfer of the rare cells C to the photocurable resin R can be realized only by light irradiation. Therefore, the operation is simple and the risk of cell loss is reduced. Moreover, the damage to the rare cell C can also be suppressed.

  Moreover, when the photocurable resin R having a visible light transmittance of 20% or more after curing is used as in the above embodiment, the rare cells C are retained in the photocurable resin R collected in the collecting step. In this state, it becomes possible to observe rare cells.

  Further, as in the above embodiment, the configuration is such that the photocurable resin R on the upper space A1 side corresponding to one surface side of the filter 105 is collected separately from the photocurable resin in the other regions, thereby being rare. Subsequent operations such as observation of cells C or analysis of rare cells C taken out from the photocurable resin R can be suitably performed.

  Moreover, it becomes possible to perform the detailed analysis which concerns on rare cells, such as a gene analysis, by including the process of collect | recovering the rare cells C separately like the said embodiment.

  As mentioned above, although embodiment of this invention was described in detail, this invention is not limited to the said embodiment, A various deformation | transformation aspect is possible. In the above embodiment, the region corresponding to one surface side of the filter 105 in the cell trapping device 100, that is, the photocurable resin R in the upper space A1 is separated from the photocurable resin in other regions such as the lower space A2. Although the case where it collects separately was demonstrated, you may collect | recover the photocurable resin R introduce | transduced in the cell capture device 100 and hardened | cured integrally. In this case, since it is considered that the photocurable resin R and the filter 105 are integrated in the cell trapping device 100, it is considered that the filter 105 is also collected together. However, since it is considered that the rare cells C are transferred to a state held by the photocurable resin R when the photocurable resin R is cured, the rare cell C is rarely removed after the filter 105 is removed from the photocurable resin R. By collecting the cells C, the cells can be suitably collected while preventing loss of rare cells.

<Example 1>
(Preparation of rare cell-containing liquid sample)
As a sample of a rare cell-containing liquid, blood containing 1000 human non-small cell lung cancer cell lines NCI-H358 per mL of blood in blood of a healthy person collected in a vacuum blood collection tube (cfDNA blood collection tube, manufactured by Streck) Samples were used. The blood used was 6 hours after blood collection. In addition, NCI-H358 used was previously fluorescently stained with CellTracker Green (manufactured by Takara Bio Inc.).

(Capture, transfer and recover cancer cells)
After attaching the filter to the cell trapping device, it was set in a CTC recovery device: MC6100 (manufactured by Hitachi Chemical Co., Ltd.), and the following experiment was performed. The filter used was a nickel thin film of 20 mm x 20 mm x 20 μm and gold-plated with a thickness of 0.2 μm. An opening (through hole) of 7.8 μm x 100 μm had an aperture ratio of 6.7%. Have in. Here, the aperture ratio refers to the area occupied by the through holes with respect to the entire area of the filter. This cell trapping device corresponds to that described in the above embodiment. The CTC recovery device includes a flow channel for introducing a blood sample and a reagent. A reservoir produced by processing a 10 mL syringe was connected to the entrance of this flow path.

  The inside of the CTC recovery apparatus was filled with distilled water, and the apparatus was primed. 7 mL of 2 mM EDTA-10.0% FBS (fetal bovine serum) -PBS (phosphate buffer, pH 7.4, manufactured by Gibco) (hereinafter also referred to as “washing solution”) is placed in the reservoir, and the lower part of the reservoir is placed. The blood sample was added so that the blood sample and the wash solution were layered.

  The CTC recovery device was activated, the blood sample and the washing solution in the reservoir were sent to the filter at a flow rate of 200 μL / min, and the cancer cells were captured by the filter. As a result, the blood in the lower reservoir layer flowed first, and then the cleaning liquid in the upper reservoir layer flowed. After confirming that the washing liquid was filled in the cell trapping device, the liquid feeding was stopped.

  Next, the liquid in the reservoir was changed to the photocurable resin R. The photocurable resin R used in the examples is polyethylene glycol dimethacrylate (14G, Shin-Nakamura Chemical Co., Ltd.), and Irgacure 2959 (manufactured by BASF) is added, mixed and dissolved as a photopolymerizable initiator. It has been made. The photocurable resin R in the reservoir is introduced into the cell trapping device at a flow rate of 200 μL / min, and the solution is sufficiently fed until the internal space is replaced by the photocurable resin R, and then the photocurable resin is fed. The liquid was stopped.

Next, the photocurable resin in the cell trapping device was irradiated with ultraviolet rays (wavelength 365 nm) for 10 seconds so that the integrated light amount was 100 mJ / cm ² to cure the photocurable resin. Thereafter, the photocurable resin inside the cell trapping device was taken out and decomposed into a photocurable resin corresponding to the upper space A1, a photocurable resin corresponding to the lower space A2, and a filter portion by tweezers.

<Example 2>
The same procedure as in Example 1 was performed except that polyethylene glycol dimethacrylate (14G, Shin-Nakamura Chemical Co., Ltd.) was replaced with polyethylene glycol diacrylate (A-600, Shin-Nakamura Chemical Co., Ltd.).

(Counting the number of cells)
The photo-curable resin corresponding to the upper space A1, the photo-curable resin corresponding to the lower space A2, and the cells remaining in the filter portion are converted into a fluorescence microscope (Axio Imager) equipped with an electric stage (ProScan II, manufactured by Prior). 2 and manufactured by Zeiss). In order to observe fluorescence derived from CellTracker Green, 38 filters (manufactured by Zeiss) were used in a fluorescence microscope. An image of the observation field was acquired, and the number of cancer cells captured by the filter was counted. Lumina Vision (manufactured by Mitani Corporation) was used for image acquisition and analysis.

  When the number of cells existing in each of the photocurable resin corresponding to the upper space A1, the photocurable resin corresponding to the lower space A2, and the filter portion was compared, It was confirmed that 90% of the cells were present. In addition, the number of cells present in the photocurable resin corresponding to the lower space A2 was zero, and it was confirmed that 10% of the total number of cells was present in the filter portion. The cells present in the filter portion are presumed to be cells that have entered the filter through-hole.

<Comparison with direct recovery from filter>
A method similar to the method described in Examples 1 and 2 above is used to directly recover cancer cells captured on a filter using a manipulator, and a photocurable resin as in Examples 1 and 2 above. The recovery success rate was compared with the case of recovery using In any case, a recovery operation was performed using a manipulator for a plurality of cancer cells. When the cancer cells captured on the filter are directly collected, the time required for collecting one cancer cell is about 10 minutes. Of the 29 cancer cells to be collected, There was one. On the other hand, as described in Examples 1 and 2, when recovering cancer cells from the photocurable resin corresponding to the upper space A1, the time required for recovering one cancer cell is about 1 minute, and All of the 30 cancer cells were successfully recovered. Thus, it has confirmed that recovery efficiency improved by performing recovery using photocurable resin.

  DESCRIPTION OF SYMBOLS 100 ... Cell capture device, 105 ... Filter, 106 ... Through-hole, 110 ... Upper member, 115 ... Lower member, 120 ... Housing, 125 ... Inflow pipe, 130 ... Inlet, 135 ... Outlet, 140 ... Outlet, A1: upper space, A2: lower space, B: buffer, C: rare cell, R: photocurable resin.

Claims (4)

A housing having an inlet for introducing liquid into the interior and an outlet for discharging liquid to the outside; and a flow path inside the housing between the inlet and the outlet. A rare cell recovery method using a cell capture device provided with a filter having a plurality of through-holes in the thickness direction,
The rare cell-containing liquid is introduced into the rare cell-containing liquid on one surface of the filter by introducing the rare cell-containing liquid from the inlet and discharging the rare cell-containing liquid through the outlet and filtering the rare cell-containing liquid through the filter. A capture step for capturing rare cells,
A buffer replacement step of filling the flow path in the cell trapping device with a buffer by introducing a buffer from the inlet;
A resin replacement step of replacing the buffer of the flow path in the cell trapping device with the photocurable resin by introducing a photocurable resin from the inlet;
A transfer step of transferring the rare cells from one surface of the filter to the photocurable resin by curing the photocurable resin in the cell trapping device;
A recovery step of taking out and recovering the photocurable resin cured in the transfer step from the cell capture device;
A method for collecting rare cells.   The method for recovering rare cells according to claim 1, wherein the photocurable resin has a visible light transmittance of 20% or more after curing.   The rare cell collection method according to claim 1, wherein in the collection step, the photocurable resin corresponding to one surface side of the filter is collected separately from the other photocurable resins.   The method for collecting rare cells according to claim 1 or 2, further comprising the step of individually collecting the rare cells from the photocurable resin taken out from the cell capture device in the collection step.

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