Rare cell capturing device and cell heterogeneity analysis method

Abstract

The invention discloses a rare cell capturing device and a cell heterogeneity analysis method, the device comprises: a sample liquid inlet for receiving a sample liquid; the first conveying channel is connected with the sample liquid inlet; the filtering area is connected with the first conveying channel and is used for filtering the sample liquid to obtain target liquid; the cell zoning and separating area is connected with the filtering area and is used for capturing rare cells from the target liquid; the second conveying channel is connected with the cell partition separation region; and the filtrate outlet is connected with the second conveying channel and is used for outputting the filtrate. The cell separation is realized, and the cell purity, the recovery rate and the sample capacity are improved. The method can be widely applied to the technical field of cell separation.

Description

Technical Field

[0001] The present invention relates to the technical field of cell separation, and in particular to a rare cell capture device and a method for analyzing cell heterogeneity. Background Art

[0002] Rare cells in blood can be used as indicators to reflect the heterogeneity between different individuals and between different tumor lesions within an individual. The counting and characteristic analysis of circulating tumor cells (CTCs) can be used as an important basis for judging the prognosis of treatment. However, the content of circulating tumor cells in blood is extremely low, about 1 / 1,000,000 of blood cells. In order to detect circulating tumor cells, it is necessary to quickly and efficiently separate and count rare cells from whole blood. Currently, there are mainly four methods, namely weirs, columns, cross-flow, and membranes, in microfluidic chips based on filtration. However, their cell sensitivity is low, it is difficult to directly separate circulating tumor cells, and there are problems such as low purity, low sample volume, and cell clogging of the membrane, resulting in low accuracy in subsequent analysis of cell heterogeneity. At the same time, it is difficult to achieve efficient recovery of target filtered cells in the chip, and the cell recovery rate is low.

[0003] In summary, the technical problems existing in the related art need to be improved. Summary of the Invention

[0004] The embodiments of the present invention provide a rare cell capture device and a method for analyzing cell heterogeneity, effectively improving cell purity, recovery rate, and sample volume.

[0005] On the one hand, the embodiments of the present invention provide a rare cell capture device, including:

[0006] A sample liquid inlet for receiving a sample liquid;

[0007] A first delivery channel connected to the sample liquid inlet;

[0008] A filtration area connected to the first delivery channel for filtering the sample liquid to obtain a target liquid;

[0009] A cell partition separation area connected to the filtration area for capturing rare cells from the target liquid;

[0010] A second delivery channel connected to the cell partition separation area;

[0011] A filtrate outlet connected to the second delivery channel for outputting the filtrate.

[0012] In some embodiments, the filtration area includes:

[0013] The tube wall of the filtration area channel;

[0014] The inlet of the filtration zone, where the inlet of the filtration zone is connected to the first conveying channel, and the inlet of the filtration zone is connected to the tube wall of the filtration zone channel by a tangent curve;

[0015] The first circular microcolumn array, which is arranged within the enclosed area of the tube wall of the filtration zone channel. The first circular microcolumn array is symmetric about the center line connecting the inlets and outlets, where the center line connecting the inlets and outlets is the line connecting the center point of the inlet of the filtration zone and the center point of the inlet of the cell separation zone. The size of each column of circular microcolumns in the first circular microcolumn array decreases in sequence;

[0016] The sample liquid channel, which is connected to the tube wall of the filtration zone channel, the inlet of the filtration zone, and the first circular microcolumn array.

[0017] In some embodiments, the cell separation zone includes:

[0018] The tube wall of the cell separation zone channel;

[0019] The inlet of the cell separation zone, where the inlet of the cell separation zone is connected to the filtration zone;

[0020] The second circular microcolumn array, which is arranged in a straight line;

[0021] The target liquid channel, which is connected to the inlet of the cell separation zone and the second circular microcolumn array;

[0022] The first layer of rounded rhombus microcolumn array, which is connected to the target liquid channel;

[0023] The first layer of side liquid channel, which is connected to the first layer of rounded rhombus microcolumn array;

[0024] The second layer of rounded rhombus microcolumn array, which is connected to the first layer of side liquid channel;

[0025] The second layer of side liquid channel, which is connected to the second layer of rounded rhombus microcolumn array;

[0026] The third layer of rounded rhombus microcolumn array, which is connected to the second layer of side liquid channel;

[0027] The outer layer of side liquid channel, which is connected to the third layer of rounded rhombus microcolumn array and the tube wall of the cell separation zone channel;

[0028] The outlet of the cell partition separation area is connected to the outer layer side liquid channel and the second delivery channel, and the outlet of the cell partition separation area is connected to the tube wall of the cell partition separation area channel by a tangent curve.

[0029] In some embodiments, the center point spacing between every two adjacent circular micro-columns in the second circular micro-column array is equal, the sizes of the circular micro-columns in the second circular micro-column array decrease in sequence, and the second circular micro-column array is used to change the moving direction of the rare cell in the target liquid channel.

[0030] In some embodiments, there is a first micropore between every two adjacent rounded rhombus micro-columns in the first layer of rounded rhombus micro-column array, there is a second micropore between every two adjacent rounded rhombus micro-columns in the second layer of rounded rhombus micro-column array, and there is a third micropore between every two adjacent rounded rhombus micro-columns in the third layer of rounded rhombus micro-column array;

[0031] The size of the second micropore is less than or equal to the size of the first micropore, the size of the third micropore is less than or equal to the size of the second micropore, and the sizes of the first micropore, the second micropore and the third micropore are all less than the size of the rare cell.

[0032] In some embodiments, the included angle between the side of the rounded rhombus micro-column in the target rounded rhombus micro-column array and the connecting line of the region center is 45 degrees;

[0033] The target rounded rhombus micro-column array includes the first layer of rounded rhombus micro-column array, the second layer of rounded rhombus micro-column array or the third layer of rounded rhombus micro-column array, and the connecting line of the region center is the connecting line between the center of the filtration area and the center of the cell partition separation area.

[0034] In some embodiments, the shape of the filtration area is the same as the shape of a water droplet dripping.

[0035] In some embodiments, the aspect ratio of the cell partition separation area is less than the aspect ratio of the filtration area.

[0036] In some embodiments, the manufacturing material of the device includes polydimethylsiloxane or plexiglass.

[0037] The beneficial effects of the present invention are as follows:

[0038] A rare cell capture device provided by an embodiment of the present invention includes a sample liquid inlet, a first delivery channel, a filtration area, a cell partition separation area, a second delivery channel, and a filtrate outlet. Among them, the sample liquid inlet is used to receive the sample liquid; the first delivery channel is connected to the sample liquid inlet; the filtration area is connected to the first delivery channel, and the sample liquid is filtered by the filtration area to obtain a target liquid; the cell partition separation area is connected to the filtration area, and rare cells are captured from the target liquid by the cell partition separation area; the second delivery channel is connected to the cell partition separation area; the filtrate outlet is connected to the second delivery channel, and the filtrate is output through the filtrate outlet, so that the rare cells in the sample liquid can be separated by the filtration area and the cell partition separation area, improving the cell purity, recovery rate, and sample capacity.

[0039] On the other hand, an embodiment of the present invention provides a method for analyzing cell heterogeneity, including the following steps:

[0040] Inject the sample liquid into the sample liquid inlet. The sample liquid flows into the filtration area after passing through the first delivery channel. The filtration area is used to filter large-area clots in the sample liquid to obtain a target liquid. The target liquid flows into the cell partition separation area through the cell partition separation area inlet. The rounded rhombus microcolumns in the cell partition separation area are used to block rare cells in the target liquid, and the cell partition separation area is used to capture the rare cells in partitions;

[0041] Inject a forward flushing liquid into the sample liquid inlet. The forward flushing liquid is used to flush the rare cells to obtain a filtrate. The filtrate includes non-target cells and waste liquid. The filtrate is discharged from the filtrate outlet after passing through the second delivery channel;

[0042] According to the flow rate of the sample liquid, perform differential analysis on the rare cells captured in partitions to obtain the relationship between the size difference of rare cells, the quantity, and the corresponding partitions at different flow rates;

[0043] According to the relationship between the size difference of rare cells, the quantity, and the corresponding partitions at different flow rates, analyze and obtain the cell heterogeneity result.

[0044] The beneficial effects of the present invention are as follows:

[0045] In an embodiment of the present invention, a sample liquid is first injected into the sample liquid inlet. After passing through the first delivery channel, the sample liquid flows into the filtration area, which is used to filter large-area clots in the sample liquid to obtain a target liquid. The target liquid flows into the cell partition separation area through the cell partition separation area inlet. Then, a forward flushing liquid is injected into the sample liquid inlet, and the forward flushing liquid is used to flush rare cells to obtain a filtrate. The filtrate is discharged from the filtrate outlet after passing through the second delivery channel. Then, according to the flow rate of the sample liquid, differential analysis is performed on the rare cells captured in the partitions to obtain the relationship between the size difference, quantity of rare cells and the corresponding partitions at different flow rates. Finally, based on the relationship between the size difference, quantity of rare cells and the corresponding partitions at different flow rates, the cell heterogeneity result is analyzed, thereby realizing cell separation and cell heterogeneity, and further improving cell purity, recovery rate, sample volume and analysis accuracy.

[0046] Other features and advantages of the present invention will be described in the following specification, and in part will be obvious from the specification, or will be understood by implementing the present invention. The objectives and other advantages of the present invention can be realized and obtained by the structures specifically pointed out in the specification and the drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the following drawings are only some embodiments of the present application. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0048] Figure 1 It is a schematic structural diagram of a rare cell capture device according to an embodiment of the present invention;

[0049] Figure 2 It is a schematic diagram of a filtration area according to an embodiment of the present invention;

[0050] Figure 3 It is a schematic diagram of an inlet of a filtration area according to an embodiment of the present invention;

[0051] Figure 4 It is a schematic diagram of a cell partition separation area according to an embodiment of the present invention;

[0052] Figure 5 It is a schematic diagram of an inlet of a cell partition separation area according to an embodiment of the present invention;

[0053] Figure 6 It is a schematic diagram of an outlet of a cell partition separation area according to an embodiment of the present invention;

[0054] Figure 7 It is a schematic diagram of a rounded rhombus microcolumn and a micropore according to an embodiment of the present invention;

[0055] Figure 8 Schematic diagram of a target rounded rhombus micro-column array according to an embodiment of the present invention;

[0056] Figure 9 Flow chart of a cell heterogeneity analysis method according to an embodiment of the present invention;

[0057] Figure 10 Schematic diagram of capturing and recovering experimental results according to an embodiment of the present invention;

[0058] Figure 11 Schematic diagram of the capturing result of rare cells without drug treatment according to an embodiment of the present invention;

[0059] Figure 12 Schematic diagram of the capturing result of rare cells after drug treatment according to an embodiment of the present invention. Detailed implementation manners

[0060] In order to make the objectives, technical solutions and advantages of the present application clearer, the present application will be further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application. When the following description refers to the accompanying drawings, unless otherwise indicated, the same numbers in different drawings represent the same or similar elements. The implementation manners described in the following exemplary embodiments do not represent all implementation manners consistent with the embodiments of the present application. They are only examples of devices and methods consistent with some aspects of the embodiments of the present application detailed in the appended claims.

[0061] It can be understood that the terms "first", "second", etc. used in the present application may be used herein to describe various concepts, but unless otherwise specified, these concepts are not limited by these terms. These terms are only used to distinguish one concept from another. For example, without departing from the scope of the embodiments of the present application, the first information may also be referred to as the second information, and similarly, the second information may also be referred to as the first information. Depending on the context, the words "if", "when" as used herein may be interpreted as "when...", "while...", or "in response to determining".

[0062] The terms "at least one", "a plurality of", "each", "any one", etc. used in the present application, at least one includes one, two or more than two, a plurality includes two or more than two, each refers to each of the corresponding plurality, and any one refers to any one of the plurality.

[0063] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terms used herein are for the purpose of describing embodiments of this application only and are not intended to limit this application.

[0064] Before elaborating on the embodiments of this application in detail, some of the nouns and terms involved in the embodiments of this application are first explained, and the nouns and terms involved in the embodiments of this application are subject to the following explanations.

[0065] Microfluidics: It integrates basic operation units such as sample preparation, reaction, separation, and detection in the processes of biological, chemical, and medical analysis onto a chip at the micron scale to automatically complete the entire analysis process.

[0066] In related technologies, specific rare cells in blood, as an indicator, reflect the heterogeneity among different individuals and among different tumor lesions within an individual. The counting and characteristic analysis of circulating tumor cells (CTCs) can be used as an important basis for breast cancer metastasis detection. However, the content of circulating tumor cells (CTCs) in blood is extremely low, approximately 1 / 1,000,000 of blood cells. To achieve CTC detection, it is necessary to establish the theoretical relationship between the drug use situation of CTCs and their physical and biological characteristics to reflect the state of CTCs, and then quickly and efficiently separate and count rare cells from whole blood through this theoretical relationship, and evaluate the cell separation situation through their distribution. Currently, microfluidic technology has become an important means for separating rare cells in blood, mainly divided into active separation and passive separation. Compared with the active separation method that uses additional force fields (electric fields, magnetic fields, sound waves, and light) to achieve cell separation, the passive separation method that relies on the structural design of the microfluidic chip and microfluidic dynamics to achieve separation has the advantages of not requiring additional force fields and additional control systems, and being simple and easy to operate, so it is widely used. Among them, the filter-type microfluidic chip design based on the physical characteristics of circulating tumor cells has a relatively low manufacturing cost and is relatively flexible to use, and can achieve the purpose of rapid detection, which is the currently commonly used separation method. Currently, there are mainly four methods, namely weir, column, cross-flow, and membrane, in the filter-based microfluidic chip. However, its cell sensitivity is poor, and few methods can directly analyze and evaluate CTCs. At the same time, it involves problems such as cell clogging of the membrane, low recovery rate, low cell purity, low cell viability, and low sample volume caused by medium yield, resulting in low accuracy of subsequent cell heterogeneity analysis and making it difficult to achieve efficient recovery of filtered circulating tumor cells in the chip.

[0067] In view of this, an embodiment of the present invention provides a rare cell capture device and a cell heterogeneity analysis method, which do not rely on the expression of tumor markers on the surface of circulating tumor cells. By utilizing the physical and biological property differences of heterogeneous circulating tumor cells, circulating tumor cells (i.e., rare cells) in whole blood are efficiently separated, captured and recovered through a filtration zone and a cell partitioning and separation zone.

[0068] The embodiments of the present application will be specifically explained below with reference to the accompanying drawings:

[0069] As Figure 1 shown, an embodiment of the present invention provides a rare cell capture device, including:

[0070] A sample liquid inlet 101 for receiving a sample liquid;

[0071] A first delivery channel 102 connected to the sample liquid inlet;

[0072] A filtration zone 103 connected to the first delivery channel for filtering the sample liquid to obtain a target liquid;

[0073] A cell partitioning and separation zone 104 connected to the filtration zone for capturing rare cells from the target liquid;

[0074] A second delivery channel 105 connected to the cell partitioning and separation zone;

[0075] A filtrate outlet 106 connected to the second delivery channel for outputting the filtrate.

[0076] In some embodiments, a rare cell capture device provided in this embodiment includes a sample liquid inlet 101, a first delivery channel 102, a filtration area 103, a cell partition separation area 104, a second delivery channel 105, and a filtrate outlet 106. A sample liquid can be received through the sample liquid inlet 101. The first delivery channel 102 is connected to the sample liquid inlet, and the sample liquid can be transported from the sample liquid inlet to the filtration area through the first delivery channel 102. The filtration area 103 is connected to the first delivery channel, and the sample liquid can be filtered through the filtration area to obtain a target liquid, where the target liquid can include a patient's blood. The channel diameter of the filtration area first increases and then decreases. The cell partition separation area 104 is connected to the filtration area, and rare cells can be captured from the target liquid through the cell partition separation area. The channel diameter of the cell partition separation area first increases and then decreases, and the channel wall of the cell partition separation area is small at both ends and large in the middle, extending horizontally with a low width-to-length ratio. The second delivery channel 105 is connected to the cell partition separation area, and the filtrate or the flushing liquid can be transported from the cell partition separation area to the filtrate outlet through the second delivery channel. The filtrate outlet 106 is connected to the second delivery channel, and the filtrate can be output through the filtrate outlet. Additionally, a microfluidic chip assembly method can be adopted to assemble the device of this embodiment with a first chip board and a second chip board. Among them, the channel grooves of the first chip board are consistent with those of the device of this embodiment, and one side of the second chip board is a smooth plane. The second chip board can be covered on the groove of the first chip board to jointly enclose a closed channel tube.

[0077] In some embodiments, as Figure 2 shown, the filtration area includes:

[0078] The channel wall of the filtration area 201;

[0079] The filtration area inlet 202, which is connected to the first delivery channel and is connected to the channel wall of the filtration area by a tangent curve;

[0080] The first circular microcolumn array 203, which is arranged within the surrounded area of the channel wall of the filtration area, is symmetric about the center line connecting the inlet and outlet, where the center line connecting the inlet and outlet is the line connecting the center point of the filtration area inlet and the center point of the cell partition separation area inlet, and the size of each column of circular microcolumns in the first circular microcolumn array decreases in sequence;

[0081] The sample liquid channel 204, which is connected to the channel wall of the filtration area, the filtration area inlet, and the first circular microcolumn array.

[0082] In some embodiments, the filtration area includes the channel wall of the filtration area 201, the filtration area inlet 202, the first circular microcolumn array 203, and the sample liquid channel 204. As Figure 3As shown, the inlet 202 of the filtration zone is connected to the first conveying channel. The inlet of the filtration zone is connected to the tube wall of the filtration zone channel by a tangent curve, which can reduce the resistance of the liquid inflow and help the sample liquid enter evenly. The inlet of the filtration zone is also the outlet of the first conveying channel. The first circular microcolumn array 203 is arranged within the enclosed area of the tube wall of the filtration zone channel. The first circular microcolumn array is symmetric about the center line connecting the inlet and outlet. The center line connecting the inlet and outlet is the line connecting the center point of the inlet of the filtration zone and the center point of the inlet of the cell separation zone. The size of each column of circular microcolumns in the first circular microcolumn array decreases in sequence. The sample liquid channel 204 is connected to the tube wall of the filtration zone channel, the inlet of the filtration zone, and the first circular microcolumn array. The sample liquid channel is formed by enclosing between the tube wall of the filtration zone channel and the first circular microcolumn array.

[0083] In some embodiments, as Figure 4 shown, the cell separation zone includes:

[0084] The tube wall 301 of the cell separation zone channel;

[0085] The inlet 302 of the cell separation zone, which is connected to the filtration zone;

[0086] The second circular microcolumn array 303, which is arranged in a straight line;

[0087] The target liquid channel 304, which is connected to the inlet of the cell separation zone and the second circular microcolumn array;

[0088] The first layer of rounded rhombus microcolumn array 305, which is connected to the target liquid channel;

[0089] The first layer of side liquid channel 306, which is connected to the first layer of rounded rhombus microcolumn array;

[0090] The second layer of rounded rhombus microcolumn array 307, which is connected to the first layer of side liquid channel;

[0091] The second layer of side liquid channel 308, which is connected to the second layer of rounded rhombus microcolumn array;

[0092] The third layer of rounded rhombus microcolumn array 309, which is connected to the second layer of side liquid channel;

[0093] The outer layer of side liquid channel 310, which is connected to the third layer of rounded rhombus microcolumn array and the tube wall of the cell separation zone channel;

[0094] The outlet 311 of the cell separation zone, which is connected to the outer layer of side liquid channel and the second conveying channel. The outlet of the cell separation zone is connected to the tube wall of the cell separation zone channel by a tangent curve.

[0095] In some embodiments, the cell partition separation region includes the cell partition separation region channel tube wall 301, the cell partition separation region inlet 302, the second circular microcolumn array 303, the target liquid channel 304, the first layer of rounded rhombus microcolumn array 305, the first layer of side liquid channels 306, the second layer of rounded rhombus microcolumn array 307, the second layer of side liquid channels 308, the third layer of rounded rhombus microcolumn array 309, the outer layer of side liquid channels 310, and the cell partition separation region outlet 311. As Figure 5 shown, the cell partition separation region inlet 302 is connected to the filtration region. Specifically, the cell partition separation region inlet is connected to the sample liquid channel in the filtration region. The cell partition separation region inlet is also the outlet of the filtration region. The second circular microcolumn array is arranged in a straight line and also in a horizontal arrangement. The target liquid channel 304 is connected to the cell partition separation region inlet and the second circular microcolumn array. The first layer of rounded rhombus microcolumn array 305 is connected to the target liquid channel. The first layer of rounded rhombus microcolumn array is composed of a number of rounded rhombus microcolumns arranged at intervals. The first layer of rounded rhombus microcolumn array and the second circular microcolumn array enclose the target liquid channel. The first layer of side liquid channels 306 is connected to the first layer of rounded rhombus microcolumn array. The second layer of rounded rhombus microcolumn array 307 is connected to the first layer of side liquid channels. The second layer of rounded rhombus microcolumn array is composed of a number of rounded rhombus microcolumns arranged at intervals. The second layer of rounded rhombus microcolumn array and the first layer of rounded rhombus microcolumn array enclose the first layer of side liquid channels. The second layer of side liquid channels 308 is connected to the second layer of rounded rhombus microcolumn array. The third layer of rounded rhombus microcolumn array 309 is connected to the second layer of side liquid channels. The third layer of rounded rhombus microcolumn array is composed of a number of rounded rhombus microcolumns arranged at intervals. The third layer of rounded rhombus microcolumn array and the second layer of rounded rhombus microcolumn array enclose the second layer of side liquid channels. The outer layer of side liquid channels 310 is connected to the third layer of rounded rhombus microcolumn array and the cell partition separation region channel tube wall. The third layer of rounded rhombus microcolumn array and the cell partition separation region channel tube wall enclose the outer layer of side liquid channels. As Figure 6 shown, the cell partition separation region outlet 311 is connected to the outer layer of side liquid channels and the second delivery channel. The cell partition separation region outlet is also the inlet of the second delivery channel. The cell partition separation region outlet is connected to the cell partition separation region channel tube wall by a tangent curve, which can seep the filtered waste liquid out of the gaps between the rounded rhombus microcolumns, and the reverse flushing liquid enters the target liquid channel through the gaps between the rounded rhombus microcolumns.

[0096] In some embodiments, the center point distance between every two adjacent circular microcolumns in the second circular microcolumn array is equal, and the sizes of the circular microcolumns in the second circular microcolumn array decrease in sequence. A circular microcolumn array with equal center point distances and a decreasing size gradient can be set at the middle position of the target liquid channel. The second circular microcolumn array is used to change the movement direction of rare cells in the target liquid channel and play a supporting role in the cell partition separation region.

[0097] In some embodiments, as Figure 7 shown, there is a first micro-hole between every two adjacent rounded rhombic micro-columns in the first layer of rounded rhombic micro-column arrays, a second micro-hole between every two adjacent rounded rhombic micro-columns in the second layer of rounded rhombic micro-column arrays, and a third micro-hole 313 between every two adjacent rounded rhombic micro-columns 312 in the third layer of rounded rhombic micro-column arrays. The size of the second micro-hole is less than or equal to the size of the first micro-hole, the size of the third micro-hole is less than or equal to the size of the second micro-hole, and the sizes of the first micro-hole, the second micro-hole, and the third micro-hole are all less than the size of rare cells. The spacing between the rounded rhombic micro-columns can be adjusted according to the size of rare cells, and the size of the rounded rhombic micro-columns and the size of the transition rounded corners can be adjusted according to the processing accuracy.

[0098] In some embodiments, as Figure 8 shown, the included angle between the side of the rounded rhombic micro-column in the target rounded rhombic micro-column array and the connecting line of the region center is 45 degrees. The target rounded rhombic micro-column array includes the first layer of rounded rhombic micro-column arrays, the second layer of rounded rhombic micro-column arrays, or the third layer of rounded rhombic micro-column arrays, and the connecting line of the region center is the connecting line between the center of the filtering area and the center of the cell partition separation area. Further, the axis of the rounded rhombic micro-column is perpendicular to the connecting line of the region center. Further, the target rounded rhombic micro-column array in the cell partition separation area can be formed by arranging a plurality of rounded rhombic micro-columns in a transverse drop and a longitudinal vertical staggered arrangement.

[0099] In some embodiments, the shape of the filtering area is the same as the shape of a water droplet dripping, and the tube wall of the filtering area channel is in the shape of a dripping water droplet.

[0100] In some embodiments, the aspect ratio of the cell partition separation area is less than the aspect ratio of the filtering area, which can avoid the problem that it is difficult for liquid to seep out due to a large aspect ratio (relative to the fluid direction). At a low aspect ratio, the liquid is more likely to enter each layer of side liquid channels through the target liquid channel (based on the wetting and hysteresis phenomena of the fluid on the rough solid surface).

[0101] In some embodiments, the manufacturing material of the device may include polydimethylsiloxane or plexiglass. Polydimethylsiloxane (PDMS) is a kind of high-molecular organosilicon compound, usually called silicone, which has the characteristics of optical transparency, inertness, non-toxicity, and non-flammability. Plexiglass has the advantages of good transparency, chemical stability, mechanical properties, weather resistance, easy dyeing, easy processing, and beautiful appearance.

[0102] The beneficial effects of implementing the embodiments of the present invention include: A rare cell capture device provided by the embodiments of the present invention includes a sample liquid inlet, a first delivery channel, a filtration area, a cell partition separation area, a second delivery channel, and a filtrate outlet. Among them, the sample liquid inlet is used to receive the sample liquid; the first delivery channel is connected to the sample liquid inlet; the filtration area is connected to the first delivery channel, and the sample liquid is filtered by the filtration area to obtain a target liquid; the cell partition separation area is connected to the filtration area, and rare cells are captured from the target liquid by the cell partition separation area; the second delivery channel is connected to the cell partition separation area; the filtrate outlet is connected to the second delivery channel, and the filtrate is output through the filtrate outlet, so that rare cells in the sample liquid can be separated by the filtration area and the cell partition separation area, improving cell purity, recovery rate, and sample capacity.

[0103] Figure 9 is an optional flowchart of a method for analyzing cell heterogeneity provided by an embodiment of the present application, Figure 9 The method in may include but is not limited to steps S401 to S404.

[0104] Step S401: Inject the sample liquid into the sample liquid inlet. The sample liquid flows into the filtration area after passing through the first delivery channel. The filtration area is used to filter large-area clots in the sample liquid to obtain a target liquid. The target liquid flows into the cell partition separation area through the cell partition separation area inlet; the rounded rhombus microcolumns in the cell partition separation area are used to block rare cells in the target liquid, and the cell partition separation area is used to partition and capture rare cells;

[0105] Step S402: Inject the forward flushing liquid into the sample liquid inlet. The forward flushing liquid is used to flush the rare cells to obtain a filtrate. The filtrate includes non-target cells and waste liquid. The filtrate is discharged from the filtrate outlet after passing through the second delivery channel;

[0106] Step S403: According to the flow rate of the sample liquid, perform differential analysis on the rare cells captured by partition to obtain the relationship between the size difference, quantity of rare cells and the corresponding partitions at different flow rates;

[0107] Step S404: Analyze and obtain the cell heterogeneity result according to the relationship between the size difference, quantity of rare cells and the corresponding partitions at different flow rates.

[0108] Steps S401 to S404 illustrated in the embodiments of the present application achieve cell separation, improving cell purity, recovery rate, and sample capacity.

[0109] In some embodiments, the sample liquid can be first injected into the sample liquid inlet. After passing through the first delivery channel, the sample liquid flows into the filtration area, where large-area clots in the sample liquid are filtered. The large-area clots are retained in the filtration area, and the target liquid is obtained. The target liquid can include patient blood. The target liquid flows into the cell partition separation area through the cell partition separation area inlet. In the cell partition separation area, rare cells in the target liquid are blocked by the rounded rhombus microcolumns. The rare cells are blocked in the first-layer side liquid channel, the second-layer side liquid channel, or the outer-layer side liquid channel in the cell partition separation area to partition and capture the rare cells. Then, the forward flushing liquid is injected into the sample liquid inlet to flush the rare cells, and the filtrate is obtained. The filtrate passes through the second delivery channel and is discharged from the filtrate outlet. The filtrate contains waste liquid and background cells (i.e., non-target cells) with smaller sizes. Then, according to the flow rate of the sample liquid, differential analysis is performed on the rare cells captured by partition to obtain the relationship between the size difference, quantity of rare cells, and the corresponding partition at different flow rates. Finally, based on the relationship between the size difference, quantity of rare cells, and the corresponding partition at different flow rates, the cell heterogeneity result is analyzed. Exemplarily, different chemotherapy drugs, such as procyanidin B2, can be used to treat human breast cancer MCF-7 cells under different action conditions to obtain heterogeneous cells. Subsequently, analysis is performed on cell proliferation activity, cytoskeleton structure, membrane proteins, etc. of the cells to obtain the correlation analysis result of the influence of the medication situation on the biological characteristics of rare cells. The medication situation includes the types of chemotherapy drugs, different medication concentrations, and different medication times. Cell proliferation activity is detected by CCK-8, MTT, etc., and cytoskeleton and membrane proteins are detected by immunofluorescence detection, etc. The heterogeneous cell solution obtained by treating with the chemotherapy drug procyanidin B2 flows into the cell partition separation area through the cell partition separation area inlet to block rare cells in the target liquid and partition and capture heterogeneous rare cells. The forward flushing liquid PBS is injected into the sample liquid inlet to flush the partition-captured heterogeneous rare cells, wash out non-target cells, and discharge the washing waste liquid through the second delivery channel from the filtrate outlet. Then, the size difference of the partition-captured human breast cancer cells is analyzed to obtain the relationship between the size difference, quantity of human breast cancer cells, and the corresponding partition, and the flow rate of the sample liquid is adjusted to obtain the relationship between the size difference, quantity of human breast cancer cells, and the corresponding partition at different flow rates, and the cell heterogeneity result of the captured human breast cancer cells is analyzed. Further, the reverse flushing liquid can be injected into the filtrate outlet, and the rare cells blocked in the first-layer side liquid channel, the second-layer side liquid channel, or the outer-layer side liquid channel are discharged from the sample liquid inlet through the reverse flushing liquid to obtain separated rare cells. The physical characteristics and biological characteristics effects of rare cells under different action conditions are different. The physical characteristic is cell size, and the biological characteristic is cell activity. There is a correlation between the physical characteristic and the biological characteristic. The medication time and concentration of breast cancer tumor cells have great heterogeneity on the physical and biological characteristics of rare cells in the blood.There is a mathematical relationship between tumor cell differences and the distribution of rare cell partitions in the cell partition separation zone. There is a correlation between rare cells under different drug treatment conditions and their distribution in the cell partition separation zone.

[0110] In some embodiments, in cell detection, 100 μl of human breast cancer cells (MCF-7) with a density of 1×10 4 cell / ml can be inoculated into a 96-well plate to obtain multiple groups of experiments to reduce errors. Incubate in an incubator (37 °C, 5% CO2) for 24 h. When the cells are completely adherent, discard the supernatant, wash 3 times with PBS, and culture with complete medium containing different concentrations of procyanidin B2 for different days. Change the culture medium with drugs every day. Take cell images by microscope imaging technology (repeated experiments, multiple groups of data), perform image processing and analysis with ImageJ software to obtain data, and then draw a cell area distribution map. At the same time, use a CCK-8 cell proliferation activity detection kit to evaluate the effect of different drug treatments on cell proliferation ability.

[0111] In some embodiments, a cell separation experiment can be carried out on this embodiment. Rabbit blood mixed with MCF-7 cells can be introduced into the sample liquid inlet at a speed of 0.2 μL / min. The sample liquid first passes through the filtration area, and blood clots and large particulate matter are retained in the filtration area. Subsequently, the target liquid enters the cell partition separation zone. During this process, single MCF-7 cells cannot pass through the micropores to achieve the capture effect. In the cell partition separation zone, MCF-7 cells are captured one by one from front to back in the target liquid channel and are captured layer by layer from the target liquid channel to each layer of the side liquid channel. In the experimental results, as Figure 10 shown, compared with the existing microfluidic rare cell separation and filtration platform in blood, this embodiment can directly separate and capture tumor cells, and the capture rate is as high as 90%, and the recovery rate is 90%. Single cells can be captured and recovered for subsequent single-cell analysis. The capture results of rare cells without drug treatment in the device partition of this embodiment are as Figure 11 shown. This embodiment can efficiently and quickly complete the separation of target cells and recover them. The capture results of rare cells after drug treatment in the device partition of this embodiment are as Figure 12 shown, showing the capture number and recovery number of the first layer of side liquid channel, the second layer of side liquid channel, and the outer layer of side liquid channel (i.e., the third layer).

[0112] The beneficial effects of implementing the embodiments of the present invention include: In the embodiments of the present invention, a sample liquid is first injected into the sample liquid inlet. After passing through the first delivery channel, the sample liquid flows into the filtration area, which is used to filter large-area clots in the sample liquid to obtain a target liquid. The target liquid flows into the cell partitioning and separation area through the cell partitioning and separation area inlet. Then, a forward flushing liquid is injected into the sample liquid inlet, and the forward flushing liquid is used to flush rare cells to obtain a filtrate. The filtrate is discharged from the filtrate outlet after passing through the second delivery channel. Then, according to the flow rate of the sample liquid, a differential analysis is performed on the rare cells captured in the partitions to obtain the relationship between the size differences, quantity of rare cells and the corresponding partitions at different flow rates. Finally, based on the relationship between the size differences, quantity of rare cells and the corresponding partitions at different flow rates, a cell heterogeneity result is analyzed, thereby realizing cell separation and cell heterogeneity, and further improving cell purity, recovery rate, sample volume and analysis accuracy.

[0113] The preferred embodiments of the embodiments of the present application have been described above with reference to the accompanying drawings, which does not limit the scope of rights of the embodiments of the present application. Any modifications, equivalent replacements, and improvements made by those skilled in the art without departing from the scope and essence of the embodiments of the present application shall be within the scope of rights of the embodiments of the present application.

Claims

1. A rare cell capture device, characterized in that, Comprising: A sample liquid inlet for receiving a sample liquid; A first conveying channel connected to the sample liquid inlet; A filtration area connected to the first conveying channel for filtering the sample liquid to obtain a target liquid; A cell partition separation area connected to the filtration area for capturing rare cells from the target liquid; A second conveying channel connected to the cell partition separation area; A filtrate outlet connected to the second conveying channel for outputting the filtrate.

2. The device according to claim 1, characterized in that, The filtration area includes: The filtration area channel tube wall; A filtration area inlet connected to the first conveying channel, and the filtration area inlet is connected to the filtration area channel tube wall by a tangent curve; A first circular micro-column array disposed within the enclosed area of the filtration area channel tube wall. The first circular micro-column array is symmetric about the center line of the inlet and outlet, and the center line of the inlet and outlet is the connection line between the center point of the filtration area inlet and the center point of the cell partition separation area inlet. The size of each column of circular micro-columns in the first circular micro-column array decreases in sequence; A sample liquid channel connected to the filtration area channel tube wall, the filtration area inlet, and the first circular micro-column array.

3. The device according to claim 1, characterized in that The cell partition separation area includes: The cell partition separation area channel tube wall; A cell partition separation area inlet connected to the filtration area; A second circular micro-column array arranged in a straight line; A target liquid channel connected to the cell partition separation area inlet and the second circular micro-column array; A first layer of rounded rhombus micro-column array connected to the target liquid channel; A first layer of side liquid channels connected to the first layer of rounded rhombus micro-column array; A second layer of rounded rhombus micro-column array connected to the first layer of side liquid channels; A second layer of side liquid channels connected to the second layer of rounded rhombus micro-column array; A third layer of rounded rhombus micro-column array connected to the second layer of side liquid channels; An outer layer of side liquid channels connected to the third layer of rounded rhombus micro-column array and the cell partition separation area channel tube wall; A cell partition separation area outlet connected to the outer layer of side liquid channels and the second conveying channel, and the cell partition separation area outlet is connected to the cell partition separation area channel tube wall by a tangent curve.

4. The device according to claim 3, characterized in that, The center point spacing between every two adjacent circular micro-columns in the second circular micro-column array is equal, the size of the circular micro-columns in the second circular micro-column array decreases in sequence, and the second circular micro-column array is used to change the movement direction of the rare cells in the target liquid channel.

5. The device according to claim 3, characterized in that, There is a first micro-pore between every two adjacent rounded rhombic micro-columns in the first layer of rounded rhombic micro-column array, a second micro-pore between every two adjacent rounded rhombic micro-columns in the second layer of rounded rhombic micro-column array, and a third micro-pore between every two adjacent rounded rhombic micro-columns in the third layer of rounded rhombic micro-column array; The size of the second micro-pore is less than or equal to the size of the first micro-pore, the size of the third micro-pore is less than or equal to the size of the second micro-pore, and the sizes of the first micro-pore, the second micro-pore and the third micro-pore are all less than the size of the rare cells.

6. The device according to claim 3, characterized in that, The included angle between the side of the rounded rhombic micro-column in the target rounded rhombic micro-column array and the connecting line of the regional center is 45 degrees; The target rounded rhombic micro-column array includes the first layer of rounded rhombic micro-column array, the second layer of rounded rhombic micro-column array or the third layer of rounded rhombic micro-column array, and the connecting line of the regional center is the connecting line between the center of the filtration area and the center of the cell partition separation area.

7. The device according to claim 1, characterized in that The shape of the filtration area is the same as the shape of a water droplet dripping.

8. The device according to claim 1, characterized in that, The aspect ratio of the cell partition separation area is less than the aspect ratio of the filtration area.

9. The device according to claim 1, wherein The manufacturing material of the device includes polydimethylsiloxane or plexiglass.

10. A method for analyzing cell heterogeneity, characterized in that, Including the following steps: Inject the sample liquid into the sample liquid inlet. The sample liquid flows into the filtration area after passing through the first delivery channel. The filtration area is used to filter large-area clots in the sample liquid to obtain the target liquid. The target liquid flows into the cell partition separation area through the cell partition separation area inlet. The rounded rhombic micro-columns in the cell partition separation area are used to block rare cells in the target liquid, and the cell partition separation area is used to partition and capture the rare cells; Inject the forward flushing liquid into the sample liquid inlet. The forward flushing liquid is used to flush the rare cells to obtain the filtrate. The filtrate includes non-target cells and waste liquid. The filtrate is discharged from the filtrate outlet after passing through the second delivery channel; According to the flow rate of the sample liquid, perform differential analysis on the partition-captured rare cells to obtain the relationship between the size difference, quantity of rare cells and the corresponding partitions at different flow rates; According to the relationship between the size difference, quantity of rare cells and the corresponding partitions at different flow rates, analyze and obtain the cell heterogeneity result.

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