High-throughput full-automatic cell separation and capture device, capture method and system

Abstract

The invention provides a high-flux full-automatic cell separation and capture device, a capture method and a system. The device comprises a storage part for storing a blood sample; the filtering part is used for filtering a flowing blood sample to obtain target cells; the lysate injection part is arranged on the upstream side of the storage part; the gas injection part is arranged on the downstream side of the filtering part, so that the filtered target cells are separated from the filtering part and are suspended in the air cushion liquid phase in the storage part; the filtering suction pump is connected with the outlet end of the filtering part; the flushing injection pump is respectively connected with the storage part and / or the filtering part through an electromagnetic three-way valve; and the controller is used for controlling the movement and direction of the fluid in the system. According to the method disclosed by the invention, the step of splitting the red blood cells is added in the filtering process, so that the residual doping of the red blood cells is remarkably eliminated, and an original gas back-flushing suspension technology is adopted in the aspect of recovering and capturing the enriched cells, so that the enriched cells can be promoted to be separated from the filtering membrane and effectively recovered.

Description

Technical Field

[0001] The present invention relates to the field of medical detection technology, and in particular to a high-throughput fully-automatic cell separation and capture device, a capture method and a system. Background Art

[0002] In the field of biological and medical research, the capture of cells with specific properties in the blood, especially rare circulating tumor cells (CTCs), is extremely important for the early diagnosis, treatment monitoring and prognosis evaluation of cancer. In the existing technology, the methods used to separate and enrich CTCs are mainly divided into three categories: based on cell physical properties, biochemical properties, and response characteristics to external force fields. Although these methods play a role in the separation and enrichment of specific types of cells, they each have limitations, and no method can perfectly meet the needs of all types of cell separation and enrichment.

[0003] Physical property separation method: separation based on physical properties of cells such as size, morphology and phenotype. Among them, cell filtration, as a physical separation and capture method, has been widely used in biological and medical research because it does not require prior biolabeling and does not affect the biological activity of cells. However, the existing cell filtration method has some obvious disadvantages, such as easy clogging of the filter membrane, cells adhering to the filter membrane after separation and enrichment are difficult to recover, the purity of the captured cells is low (especially the high degree of impurity of red blood cells), and it is usually manual operation, and the separation and enrichment process is cumbersome, time-consuming and prone to errors.

[0004] Biochemical characteristic separation method: using biochemical markers on the cell surface, such as epithelial cell adhesion molecule (EpCAM), to separate and enrich cells through immunomagnetic beads or specific antibodies. Although this method has shown certain effects in capturing specific types of CTCs, it is difficult to capture CTCs that have undergone epithelial-mesenchymal transition (EMT) because they may no longer express EpCAM.

[0005] Force field response characteristic separation method: cells are separated by applying an external force field (such as centrifugal force, electric field, magnetic field, etc.). These methods can improve the capture efficiency of CTCs to a certain extent, but often require complex equipment and operations, and may cause damage to cells.

[0006] In practical applications, although existing technologies have made some progress in capturing CTCs, they still have the following shortcomings: 1. Capture efficiency and purity: Existing technologies often find it difficult to achieve high efficiency and high purity requirements when capturing CTCs, especially when processing large amounts of blood samples. How to improve capture efficiency and reduce interference from non-target cells remains a challenge.

[0007] 2. Maintaining cell activity: During the capture and separation process, how to maintain the activity and functional status of CTCs for subsequent culture and analysis is a problem that the existing technology needs to solve.

[0008] 3. Operational complexity and cost: Many existing technologies require complex operations, expensive instruments and biological reagents, which limits their widespread promotion in clinical applications.

[0009] 4. Standardization and automation: The detection method of CTCs has not yet been standardized in clinical practice, and the degree of automation of existing technologies still needs to be improved to meet the needs of high-throughput detection.

[0010] Therefore, developing a new CTCs capture technology that can overcome the above-mentioned shortcomings and achieve high-throughput, high-efficiency, high-purity and easy-to-operate CTCs capture has important clinical significance for promoting early diagnosis and personalized treatment of cancer. Summary of the invention

[0011] The present invention provides a high-throughput fully automatic cell separation and capture device, capture method and system, which are used to solve the defects in the prior art, such as easy clogging of the filter membrane, difficulty in recovering cells adhering to the filter membrane after separation and enrichment, low purity of captured cells, high degree of red blood cell contamination, and usually manual operation, and the separation and enrichment process is cumbersome, time-consuming and error-prone.

[0012] According to a first aspect of the present invention, a high-throughput fully automatic cell separation and capture device is provided, comprising: a storage part, wherein a receiving chamber is formed inside, and the receiving chamber is used to store a blood sample; a filtering part, arranged at the downstream side of the storage part, and used to filter the blood sample flowing through to obtain target cells, wherein the filtering at least includes a separation process and an enrichment process of the blood sample; a lysate injection part, arranged at the upstream side of the storage part, and used to store and provide red blood cell lysate for cleaning the receiving chamber; a gas injection part, arranged at the downstream side of the filtering part, and used to input gas from the downstream side of the filtering part, so that the filtered target cells are separated from the filtering part and suspended in the gas cushion liquid phase in the storage part; a filtration suction pump, connected to the outlet end of the filtration part, and used to provide power for the filtration of the blood sample and collect filtrate; a flushing injection pump, respectively connected to the storage part and / or the filtration part through an electromagnetic three-way valve, and used to provide power and flushing liquid for forward and reverse flushing of the blood sample; and a controller, used to control the movement of the filtration suction pump, the flushing injection pump and the electromagnetic three-way valve, thereby controlling the movement and direction of the fluid in the system.

[0013] According to one embodiment of the present invention, the filtering part includes: a fixing unit, which is arranged on the downstream side of the storage part and connected to the storage part, and has a filtering cavity connected to the accommodating cavity formed therein; a microporous sieve, which is arranged in the filtering cavity and is used to filter the blood sample flowing through.

[0014] Specifically, this embodiment provides an implementation of a filter unit.

[0015] According to one embodiment of the present invention, the fixing unit includes: a first connecting tube connected to the storage part; a second connecting tube connected to an end of the first connecting tube away from the storage part, and the filter cavity is formed inside the first connecting tube and the second connecting tube; a first sealing ring and a second sealing ring are respectively arranged at the connection between the first connecting tube and the second connecting tube, and the microporous screen is arranged between the first sealing ring and the second sealing ring.

[0016] Specifically, this embodiment provides an implementation of a fixing unit.

[0017] According to one embodiment of the present invention, the diameter of the effective filtration area of ​​the microporous sieve is greater than or equal to 1 mm, and the surface of the microporous sieve is provided with filtration holes with a number ranging from 50K to 500K, and the diameter of the filtration holes is between 5 and 15 microns.

[0018] Specifically, this embodiment provides an implementation of a microporous sieve.

[0019] According to one embodiment of the present invention, it also includes: a first three-way valve, arranged between the lysis solution injection part and the storage part, for realizing the path switching when the flushing solution is added and the red blood cell lysis solution is pre-loaded in the forward cleaning pipeline; a second three-way valve, arranged between the gas injection part and the filtering part, for realizing the path switching of the flushing solution and the gas introduction.

[0020] Specifically, this embodiment provides an implementation of a first three-way valve and a second three-way valve.

[0021] According to one embodiment of the present invention, the filtration suction pump is arranged on the downstream side of the filtration section, and is used to provide power for the filtration of the blood sample and collect the filtrate; the flushing injection pump is arranged on the downstream side of the filtration section, and is used to provide power and flushing fluid for the reverse flushing of the filtration section, and / or, the flushing injection pump is arranged on the upstream side of the storage section, and is used to provide power and flushing fluid for the forward flushing of the storage section.

[0022] Specifically, this embodiment provides an implementation of a filtration suction pump and a flushing injection pump.

[0023] According to the second aspect of the present invention, a capture method of the above-mentioned high-throughput fully-automatic cell separation and capture device is provided, comprising: a preloading step, loading red blood cell lysing solution into the pipeline between the first three-way valve and the storage part through the lysing solution injection part, and loading the blood sample to be filtered into the storage part; A control parameter setting step, inputting the control parameters of the initial sample volume, the filtration suction pump and the flushing injection pump through the user interface of the control program, wherein the control parameters at least include a flow rate parameter and a flow rate parameter; an automatic filtering step, wherein the blood sample is filtered through a filter unit under the control of a controller, and target cells are obtained in the filter unit, wherein after the blood sample passing through the filter unit reaches a first set amount, the filter unit is flushed with a second set amount of flushing liquid, and the first set amount is greater than the second set amount; An automatic lysis step, under the control of the controller, the flushing injection pump drives the red blood cell lysis solution into the storage part to lyse the red blood cells retained in the storage part for a set time; An automatic flushing step, during the filtering process, after the blood sample passing through the filtering part reaches a first set amount, the filtering part is backwashed with a second set amount of flushing liquid to prevent the microporous screen from being blocked, the first set amount being greater than the second set amount, and after the red blood cell lysis is completed, the flushing injection pump and the filtration suction pump will work alternately to complete the washing of the enriched cells; The suspension step is to introduce gas from the downstream side of the filter section so that the target cells are separated from the microporous sieve in the filter section, suspended in the air cushion liquid phase of the storage section, and recovered by a pipette.

[0024] According to one embodiment of the present invention, before the suspending step, the method further comprises: The storage part after red blood cell lysis is flushed a set number of times. During the flushing process, the filter part is flushed from the downstream side of the filter part with a second set amount of flushing liquid and enters the storage part, and the storage part is flushed from the upstream side of the storage part with a third set amount of flushing liquid, wherein the third set amount is between the second set amount and the first set amount.

[0025] Specifically, this embodiment provides an implementation method before the suspension step.

[0026] According to the third aspect of the present invention, a high-throughput cell separation and capture system is provided, comprising: a controller for setting, controlling and displaying the motion parameters and motion states of a filtration suction pump, a flushing injection pump and an electromagnetic three-way valve, thereby controlling the movement and direction of the fluid in the system to complete the filtration of the sample and the cleaning of the enriched cells; the above-mentioned high-throughput fully automatic cell separation and capture device, or, when separating cells, using the capture method of the above-mentioned high-throughput fully automatic cell separation and capture device; wherein the controller is connected to the filtration suction pump, the flushing injection pump and the electromagnetic three-way valve, and is used to automatically filter the blood sample in the storage unit, lyse the red blood cells and flush them to obtain high-purity target cells.

[0027] The above one or more technical solutions in the present invention have at least one of the following technical effects: a high-throughput fully automatic cell separation and capture device, capture method and system provided by the present invention significantly eliminates residual impurities of red blood cells by adding a red blood cell lysis step in the filtration process, and adopts a one-button fully automatic cell filtration enrichment control technology, so that the separation and enrichment process of target cells is automatically controlled by a computer program, which significantly reduces the workload and errors of manual operations. In terms of enriched cell recovery and capture, an original gas recoil suspension technology is adopted, which can promote the separation and effective recovery of enriched cells from the filter membrane, and can separate rare circulating tumor cells from blood samples according to their size and deformation characteristics, and a corresponding separation and enrichment operation process has been developed.

[0028] Furthermore, in the related art, the capture of cells with specific properties in the blood (such as relatively rare circulating tumor cells) plays a very important role and significance in the fields of biology and medical research. Based on the micro-sieve technology, the present invention realizes the full automation of the blood cell separation and capture device, which is used to quickly separate and enrich rare circulating tumor cells in blood samples. In addition to the loading of the whole blood sample and the extraction of the target cells after enrichment and separation, each process including blood filtration, red blood cell lysis and repeated washing is automatically completed under the control of a computer program. The present invention is compatible with downstream cell analysis processes such as standard cytopathological diagnosis and amplification culture for the rare cell capture process. The blood cell automatic separation and enrichment device of the present invention can complete the processing of 3 ml of whole blood samples within 25 minutes. And it achieves the active capture of more than 90% of the target cells, the removal of more than 99.99% of the white blood cells and 100% of the red blood cells. Compared with the existing manual or semi-automatic capture procedures, the separation and enrichment of rare cells in blood samples through a fully automated procedure will be more efficient, consistent and reliable in clinical environments. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0030] Figure 1 This is one of the schematic diagrams of the arrangement relationship of the high-throughput fully-automatic cell separation and capture device provided by the present invention.

[0031] Figure 2 This is the second schematic diagram of the arrangement relationship of the high-throughput fully-automatic cell separation and capture device provided by the present invention.

[0032] Figure 3 It is a schematic diagram of the structural relationship of the filtration part in the high-throughput fully-automatic cell separation and capture device provided by the present invention.

[0033] Figure 4 This is one of the process relationship schematic diagrams of the capture method of the high-throughput fully automatic cell separation and capture device provided by the present invention.

[0034] Figure 5 This is the second schematic diagram of the process relationship of the capture method of the high-throughput fully automatic cell separation and capture device provided by the present invention.

[0035] Reference numerals: 10. Storage department; 20. Filtering unit; 21. Fixing unit; 211. First connecting pipe; 212. Second connecting pipe; 213. First sealing ring; 214. Second sealing ring; 22. Microporous sieve; 30. Lysis solution injection part; 40. Gas injection unit; 50. Filter suction pump; 60. Flush the injection pump; 70. First three-way valve; 80. Second three-way valve; 90. Solenoid three-way valve; 100. Controller. DETAILED DESCRIPTION

[0036] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0037] In the description of the embodiments of the present invention, it should be noted that the terms "center", "longitudinal", "lateral", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside" and the like indicate positions or positional relationships based on the positions or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the embodiments of the present invention and simplifying the description, and do not indicate or imply that the devices or elements referred to must have a specific orientation, be constructed and operate in a specific orientation, and therefore cannot be understood as limitations on the embodiments of the present invention. In addition, the terms "first", "second", and "third" are used for descriptive purposes only and cannot be understood as indicating or implying relative importance.

[0038] The present invention will be described in detail below in conjunction with specific implementation modes.

[0039] In some specific embodiments of the present invention, Figures 1 to 3 As shown, the present invention provides a high-throughput fully automatic cell separation and capture device, comprising: a storage part 10, wherein a receiving chamber is formed inside the storage part 10, and the receiving chamber is used to store a blood sample; a filtering part 20, which is arranged at the downstream side of the storage part 10, and is used to filter the blood sample flowing through to obtain target cells, and the filtering at least includes a separation process and an enrichment process of the blood sample; a lysing solution injection part 30, which is arranged at the upstream side of the storage part 10, and is used to store and provide a red blood cell lysing solution for cleaning the receiving chamber; a gas injection part 40, which is arranged at the downstream side of the filtering part 20, and is used to input gas from the downstream side of the filtering part 20 body, so that the filtered target cells are separated from the filter part 20 and suspended in the air cushion liquid phase in the storage part 10; the filtration suction pump 50 is connected to the outlet end of the filtration part 20, and is used to provide power for the filtration of the blood sample and collect the filtrate; the flushing injection pump 60 is connected to the storage part 10 and / or the filtration part 20 through the electromagnetic three-way valve 90, and is used to provide power and flushing liquid for the forward and reverse flushing of the blood sample; the controller 100 is used to control the movement of the filtration suction pump 50, the flushing injection pump 60 and the electromagnetic three-way valve 90, thereby controlling the movement and direction of the fluid in the system.

[0040] It should be noted that the present invention realizes full automation of blood cell separation and capture by providing a blood cell separation and capture device and a controller 100 connected to the lysate injection part 30, the gas injection part 40, the filter suction pump 50 and the flushing injection pump 60, which solves the problem that the traditional manual filtration separation and enrichment method is a very cumbersome and error-prone process, and the new system automatically handles all time-consuming operation steps, including sample filtration, red blood cell lysis and reciprocating flushing. The operator only needs to add the blood sample to the separation and capture device, and after running the automatic enrichment and separation program, the target cells for enrichment and separation can be extracted, realizing a one-button "sample in, result out" automatic cell separation and enrichment process.

[0041] Furthermore, in the actual application of the present invention, by using an automatic system and an optimization process, the experimental verification results show that the average target cell recovery rate obtained from 3 ml of whole blood samples is >90%, the cell survival rate is >98.6%, and the impurity level of only about 300 white blood cells per ml of whole blood samples. The present invention can separate and enrich the target cells according to their size and deformation characteristics without prior biomarkers, and can efficiently separate and capture biologically active circulating tumor cells from the patient's blood, which is very suitable for liquid biopsy, opening a new window for early detection of cancer metastasis and recurrence, real-time monitoring of treatment effects, and screening of personalized therapeutic drugs.

[0042] In addition, the air cushion liquid phase refers to a liquid medium formed in the filter cavity of the filter part 20 during the blood sample filtering process.

[0043] In some possible embodiments of the present invention, the filter unit 20 includes: a fixing unit 21, which is arranged on the downstream side of the storage unit 10 and connected to the storage unit 10, and has a filter cavity connected to the accommodating cavity formed therein; a microporous sieve 22, which is arranged in the filter cavity and is used to filter the blood sample flowing through.

[0044] Specifically, this embodiment provides an implementation of the filter part 20 , which achieves filtering of the blood sample flowing through by providing a microporous sieve 22 , and the fixing unit 21 provides a fixed position for the microporous sieve 22 .

[0045] In a possible embodiment, the fixing unit 21 is connected to the storage unit 10 via a pipeline.

[0046] In some possible embodiments of the present invention, the fixing unit 21 includes: a first connecting tube 211, connected to the storage part 10; a second connecting tube 212, connected to an end of the first connecting tube 211 away from the storage part 10, and a filter cavity is formed inside the first connecting tube 211 and the second connecting tube 212; a first sealing ring 213 and a second sealing ring 214, respectively arranged at the connection between the first connecting tube 211 and the second connecting tube 212, and the microporous screen 22 is arranged between the first sealing ring 213 and the second sealing ring 214.

[0047] Specifically, this embodiment provides an implementation of a fixing unit 21, which provides a fixed position for the microporous sieve 22 through the arrangement of the first connecting tube 211 and the second connecting tube 212, and the arrangement of the first sealing ring 213 and the second sealing ring 214 realizes sealing. Through this design, the microporous sieve 22, the first sealing ring 213 and the second sealing ring 214 can be easily loaded and fixed in the first connecting tube 211 and the second connecting tube 212 for blood filtration. The microporous sieve 22 can also be easily removed from the filter part 20 for replacement or cell staining analysis. Except for the silicon microsieve and the silicone gasket, the rest of the parts of the entire device are made of engineering plastic polyethylene through precision processing.

[0048] In a possible embodiment, the first connecting pipe 211 and the second connecting pipe 212 are threadedly connected.

[0049] In some possible embodiments of the present invention, the diameter of the effective filtering area of ​​the microporous sieve 22 is greater than or equal to 1 mm, and the surface of the microporous sieve 22 is provided with filtering holes with a number ranging from 50K to 500K, and the diameter of the filtering holes is between 5 and 15 microns.

[0050] Specifically, this embodiment provides an implementation of a microporous sieve 22. The setting of the microporous sieve 22 enables the acquisition of target cells in a blood sample and improves the acquisition rate of target cells. The target cells are specific cells related to tumors.

[0051] It should be noted that the liquid passes through the microporous screen 22 in the form of axial flow, thereby achieving a high-throughput cell separation and capture effect.

[0052] In a possible embodiment, the present invention adopts a silicon micromachining method to produce a silicon microsieve filter membrane with uniform pore size, namely, a microporous sieve 22, and performs an anti-adhesion treatment on its surface, which can effectively reduce the clogging of the silicon microsieve filter membrane by blood samples.

[0053] In a possible embodiment, the micro-mesh 22 of the present invention is provided with about 90,000 micro-mesh holes, and the hole diameter is 8 mm.

[0054] In a possible embodiment, the micro-mesh 22 is soaked in the anti-stick coating solution for at least 12 hours before use.

[0055] In some possible embodiments of the present invention, it also includes: a first three-way valve 70, which is arranged between the lysis solution injection part 30 and the storage part 10, and is used to realize the path switching when the flushing solution is added and the red blood cell lysis solution is pre-loaded in the forward cleaning pipeline; a second three-way valve 80, which is arranged between the gas injection part 40 and the filtration part 20, and is used to realize the path switching of the flushing solution and gas introduction.

[0056] Specifically, this embodiment provides an implementation of a first three-way valve 70 and a second three-way valve 80. By setting the first three-way valve 70, switching between two paths of blood sample addition and red blood cell lysate addition is achieved, and the second three-way valve 80 achieves switching between blood sample filtration and gas introduction.

[0057] In a possible embodiment, both the first three-way valve 70 and the second three-way valve 80 are electrically controlled valve bodies.

[0058] In a possible embodiment, both the first three-way valve 70 and the second three-way valve 80 are manual valve bodies.

[0059] In a possible embodiment, both the first three-way valve 70 and the second three-way valve 80 are connected to the controller 100 .

[0060] In some possible embodiments of the present invention, the filtration suction pump 50 is arranged on the downstream side of the filtration section 20 to provide power for the filtration of the blood sample and to collect the filtrate; the flushing injection pump 60 is arranged on the downstream side of the filtration section 20 to provide power and flushing fluid for the reverse flushing of the filtration section 20, and / or the flushing injection pump 60 is arranged on the upstream side of the storage section 10 to provide power and flushing fluid for the forward flushing of the storage section 10.

[0061] Specifically, this embodiment provides an implementation of a filtration suction pump 50 and a flushing injection pump 60. The filtration suction pump 50 is connected to the downstream side of the filter part 20 to provide power for the flow of the blood sample, and the flushing injection pump 60 realizes flushing of the device itself.

[0062] In a possible embodiment, the flush syringe pump 60 pump and all fluid paths are pre-filled with flushing fluid prior to sample processing.

[0063] In some possible embodiments of the present invention, it further includes: an electromagnetic three-way valve 90, and the flushing injection pump 60 is connected to the filter part 20 and the storage part 10 respectively through the electromagnetic three-way valve 90 to provide flushing power and flushing liquid to the filter part 20 and the storage part 10.

[0064] Specifically, this embodiment provides an implementation of an electromagnetic three-way valve 90 . By providing the electromagnetic three-way valve 90 , the flushing injection pump 60 can switch the cleaning paths in the filter part 20 and the storage part 10 .

[0065] In some specific embodiments of the present invention, Figure 4 and Figure 5 As shown, this solution provides a capture method of the above-mentioned high-throughput fully automatic cell separation and capture device, comprising: A pre-loading step, in which a red blood cell lysing solution is loaded into the pipeline between the first three-way valve 70 and the storage part 10 through the lysing solution injection part 30, and a blood sample to be filtered is loaded into the storage part 10; A control parameter setting step, inputting the control parameters of the initial sample volume, the filtration suction pump 50 and the flushing injection pump 60 through the user interface of the control program, wherein the control parameters at least include a flow rate parameter and a flow rate parameter; An automatic filtering step, in which the blood sample is filtered through the filter unit 20 under the program control of the controller 100, and target cells are obtained in the filter unit 20, wherein after the blood sample passing through the filter unit 20 reaches a first set amount, the filter unit 20 is flushed with a second set amount of flushing liquid, and the first set amount is greater than the second set amount; Automatic lysis step: under the control of the controller 100, the flushing injection pump 60 drives the red blood cell lysis solution into the storage part 10, and lyses the red blood cells retained in the storage part 10 for a set time; Automatic flushing step: during the filtering process, after the blood sample passing through the filter part 20 reaches a first set amount, the filter part 20 is backwashed with a second set amount of flushing liquid to prevent the microporous screen 22 from being blocked. The first set amount is greater than the second set amount. After the red blood cell lysis is completed, the flushing injection pump 60 and the filter suction pump 50 will work alternately to complete the washing of the enriched cells; In the suspension step, gas is introduced from the downstream side of the filter unit 20 to separate the target cells from the microporous sieve 22 in the filter unit 20, suspend them in the air cushion liquid phase of the storage unit 10, and recover them by a pipette.

[0066] It should be noted that the target cells are suspended in the air cushion liquid phase of the storage part 10 to obtain the best cell separation and capture effect.

[0067] In a possible embodiment, the gas injection part 40 is a syringe, so in the pre-installation step, the gas injection part 40 needs to be pre-loaded with air.

[0068] In a possible embodiment, the first set amount is 2 ml, and the second set amount is 0.5 ml.

[0069] In a possible embodiment, the duration is set to 10 minutes.

[0070] In a possible embodiment, during the enrichment process, the controller 100 controls the operation of the lysate injection unit 30, the gas injection unit 40, the filtration suction pump 50, the flushing injection pump 60 and the three solenoid valves. The control program includes a user interface for setting the control parameters of the solenoid valve and the injection pump, performing start and stop control, and displaying the current progress and status of the enrichment process. Under the control of the program, the system will perform a series of steps such as filtration, backwashing and top flushing to remove a large number of red blood cells and white blood cells in the sample and separate target specific cells (such as CTCs). In addition, the system will always ensure that the target cells are immersed in the flushing solution to maintain their biological activity.

[0071] In a possible embodiment, in the filtration step, for the washing solution, in order to reduce the non-specific binding of whole blood sample cells to the filter device, a solution containing albumin is used as the washing solution. The washing solution is a mixture of 2mM disodium ethylenediaminetetraacetic acid (EDTA) and 1x Dulbecco's phosphate buffered saline (DPBS), with 0.5% (weight ratio) bovine serum albumin (BSA) added. For every 50 ml of washing solution, 250 mg of bovine serum albumin and 37.22 mg of EDTA are dissolved in 50 ml of DPBS. After dissolution, it is filtered to remove any insoluble impurities and particles. The albumin-based washing solution needs to be prepared on the day of the experiment to maintain its effectiveness. Because if the washing solution is not used within 24 hours after preparation or stored at 4°C for a long time, the effect of the protein will deteriorate exponentially.

[0072] Furthermore, a set amount of red blood cell lysis solution is pre-installed into the connecting pipeline through the first three-way valve.

[0073] In a possible embodiment, in the pre-loading step, for the part of the red blood cell lysate, in order to eliminate the contamination of the target captured cells by red blood cells (RBCs), the red blood cells need to be lysed during the enrichment process. A 10-fold concentration of red blood cell lysate is prepared from 0.15M ammonium chloride, 0.1M sodium bicarbonate, 10mM EDTA and deionized water. The prepared red blood cell lysate is usually stored in a refrigerator at 4°C and diluted to a 1x concentration before use. Like the flushing (buffer) solution, the red blood cell lysate is filtered through a 0.2μm filter membrane before use to remove possible precipitates.

[0074] In a possible embodiment, in the suspension step, the target cells are separated from the microporous mesh 22 in the filter part 20 and suspended in the air cushion liquid phase at the bottom side of the storage part 10 .

[0075] In some possible embodiments of the present invention, before the suspending step, the method further comprises: The storage part 10 is flushed a set number of times after the red blood cell lysis. During the flushing process, the filter part 20 is flushed from the downstream side of the filter part 20 with a second set amount of flushing liquid and enters the storage part 10, and is flushed from the upstream side of the storage part 10 with a third set amount of flushing liquid, wherein the third set amount is between the second set amount and the first set amount.

[0076] Specifically, this embodiment provides an implementation method before the suspension step, which removes most of the white blood cells and lysed red blood cell debris by washing for a set number of times.

[0077] In a possible embodiment, the third set amount is 1 ml.

[0078] In a possible embodiment, the number of times is set to be greater than or equal to 4 times.

[0079] In some specific embodiments of the present invention, the present invention provides a high-throughput cell separation and capture system, comprising: a controller 100, used to set, control and display the motion parameters and motion states of the filtration suction pump 50, the flushing injection pump 60 and the electromagnetic three-way valve 90, so as to control the movement and direction of the fluid in the system to complete the filtration of the sample and the cleaning of the enriched cells; the above-mentioned high-throughput fully automatic cell separation and capture device, or, when separating cells, the capture method of the above-mentioned high-throughput fully automatic cell separation and capture device is adopted; wherein the controller 100 is connected to the filtration suction pump 50, the flushing injection pump 60 and the electromagnetic three-way valve 90, and is used to automatically filter the blood sample in the storage part 10, lyse the red blood cells and flush them to obtain high-purity target cells.

[0080] In a possible embodiment, the controller 100 is respectively connected to the lysis solution injection part 30, the gas injection part 40, the filtration suction pump 50 and the flushing injection pump 60. In this embodiment, the lysis solution injection part 30, the gas injection part 40, the filtration suction pump 50 and the flushing injection pump 60 are all electronically controlled components.

[0081] In a possible embodiment, the filter pump can be set to different flow rates according to actual needs to optimize the process.

[0082] It should be noted that due to the limited number of CTCs in the patient's blood, high recovery and high purity separation and capture are essential for accurate CTC counting and downstream analysis. Therefore, improving recovery and reducing other cell contamination may be the most basic goal of any CTC enrichment and capture device. In order to reduce the loss of CTCs, minimize blood cell contamination and maximize the recovery rate of CTCs, several key technologies have been developed for automated CTC enrichment systems.

[0083] The first key technology is fixed-point sample handling. Sample handling is confined to a very small and relatively fixed space between the bottom of the sample storage / handler and the upper part of the connected filtration device. This will help avoid the loss of CTCs during the enrichment process because there is no sample transfer step involved. During the enrichment process, when the sample is transferred from one device to another, or even from one part of the device to another, unnecessary loss of CTCs may occur.

[0084] The second key technology is to precisely control the liquid level above the microsieve to prevent accidental dehydration of the microsieve, which is critical to the operation of the enrichment process. During filtration and rinsing, if the top of the microsieve is dehydrated, air will be sucked through the microsieve, forming a huge surface tension that can lead to target cell loss. Therefore, a thin layer of liquid should always be maintained above the microsieve. On the other hand, if the residual liquid level is too high, the dead space capacity of the rinse will be too much, and more rinses may be required to remove blood cells and lysis products.

[0085] The third key technology is to pre-load the red blood cell (RBC) lysis solution into the top flush line of the enrichment unit. By pre-loading, the system automatically adds lysis to perform RBC lysis during the enrichment process. The purpose of RBC lysis is to completely remove the red blood cells that remain adhered to the sample reservoir / processor and tubing after filtration. After the blood sample is filtered, a large number of red blood cells remain attached to the fluid channel and sample reservoir. If not removed, they may fall back into the solution again during the backwash / forward flush process, reducing the purity of the recovered target cells.

[0086] The fourth key technology is to release CTCs from the microsieve by air assistance. Unlike existing CTC filtration systems, one of the outstanding features of the enrichment system of the present invention is that the enriched cancer cells can be effectively released from the microsieve and suspended in the air cushion liquid phase for extraction. This feature will greatly facilitate the downstream analysis of captured CTCs. To this end, the present invention has developed an air-assisted recovery technology to maximize the extraction of captured CTCs from the microsieve. Usually after filtering and washing, the target cells will be captured on the microsieve. In order to release these cells from the microsieve, the backwashing can resuspend the cells in the solution. However, if the cell suspension is not extracted and collected in time, some target cells may settle and reattach to the microsieve. In addition, it is very difficult to completely extract the cell suspension from the sample reservoir because the pipette tip must be kept at a certain distance from the microsieve to avoid accidentally damaging the microsieve. Through air-assisted recovery, the cell suspension can be collected in full, thereby minimizing the loss of target cells and maximizing the recovery rate.

[0087] In the description of the embodiments of the present invention, it should be noted that, unless otherwise clearly specified and limited, the terms "connected" and "connection" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection or an indirect connection through an intermediate medium. For ordinary technicians in this field, the specific meanings of the above terms in the embodiments of the present invention can be understood according to specific circumstances.

[0088] In the embodiments of the present invention, unless otherwise clearly specified and limited, the first feature being "above" or "below" the second feature may mean that the first and second features are in direct contact, or the first and second features are in indirect contact through an intermediate medium. Moreover, the first feature being "above", "above" and "above" the second feature may mean that the first feature is directly above or obliquely above the second feature, or simply means that the first feature is higher in level than the second feature. The first feature being "below", "below" and "below" the second feature may mean that the first feature is directly below or obliquely below the second feature, or simply means that the first feature is lower in level than the second feature.

[0089] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "mode", "specific mode", or "some modes" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or mode are included in at least one embodiment or mode of the embodiment of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or mode. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or modes in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or modes described in this specification and the features of the different embodiments or modes, without contradiction.

[0090] Finally, it should be noted that the above embodiments are only used to illustrate the present invention, rather than to limit the present invention. Although the present invention is described in detail with reference to the embodiments, it should be understood by those skilled in the art that various combinations, modifications or equivalent substitutions of the technical solutions of the present invention do not depart from the spirit and scope of the technical solutions of the present invention, and should be included in the scope of the claims of the present invention.

Claims

1. A high-throughput fully automatic cell separation and capture device, characterized in that: include: A storage portion (10) having a receiving cavity formed therein, the receiving cavity being used to store a blood sample; A filtering unit (20), arranged at the downstream side of the storage unit (10), for filtering the blood sample flowing through to obtain target cells, wherein the filtering at least includes a separation process and an enrichment process of the blood sample; A lysing solution injection portion (30), disposed at the upstream side of the storage portion (10), and used for storing and providing red blood cell lysing solution for cleaning the containing chamber; a gas injection section (40) disposed at the downstream side of the filter section (20) and used to input gas from the downstream side of the filter section (20) so that the filtered target cells are separated from the filter section (20) and suspended in the gas cushion liquid phase in the storage section (10); A filtration suction pump (50), connected to the outlet end of the filtration unit (20), for providing power for filtering the blood sample and collecting filtrate; The flushing syringe pump (60) is respectively connected to the storage part (10) and / or the filtering part (20) through an electromagnetic three-way valve (90), and is used to provide power and flushing fluid for forward and reverse flushing of the blood sample; The controller (100) is used to control the movement of the filtering suction pump (50), the flushing injection pump (60) and the electromagnetic three-way valve (90), thereby controlling the movement and direction of the fluid in the system.

2. The high-throughput fully-automatic cell separation and capture device according to claim 1, characterized in that: The filter unit (20) comprises: A fixing unit (21) is arranged at the downstream side of the storage portion (10), is connected to the storage portion (10), and has a filter cavity formed therein that is in communication with the accommodating cavity; A microporous sieve (22) is arranged in the filter cavity and is used to filter the blood sample flowing through the sieve.

3. The high-throughput fully-automatic cell separation and capture device according to claim 2, characterized in that: The fixing unit (21) comprises: A first connecting pipe (211) connected to the storage portion (10); a second connecting tube (212) connected to an end of the first connecting tube (211) away from the storage portion (10), and the first connecting tube (211) and the second connecting tube (212) form the filter cavity inside; A first sealing ring (213) and a second sealing ring (214) are respectively arranged at the connection between the first connecting tube (211) and the second connecting tube (212), and the microporous screen (22) is arranged between the first sealing ring (213) and the second sealing ring (214).

4. The high-throughput fully-automatic cell separation and capture device according to claim 2, characterized in that: The diameter of the effective filtering area of ​​the microporous sieve (22) is greater than or equal to 1 millimeter, and the surface of the microporous sieve (22) is provided with filtering holes with a number ranging from 50K to 500K, and the diameter of the filtering holes is between 5 and 15 micrometers.

5. The high-throughput fully-automatic cell separation and capture device according to any one of claims 1 to 4, characterized in that: Also includes: A first three-way valve (70) is disposed between the lysing solution injection portion (30) and the storage portion (10) and is used to implement the switching of the passages when the flushing solution is added and the red blood cell lysing solution is pre-loaded in the forward washing pipeline; A second three-way valve (80) is provided between the gas injection portion (40) and the filter portion (20) and is used to switch the passages for introducing the flushing liquid and the gas.

6. The high-throughput fully-automatic cell separation and capture device according to any one of claims 1 to 4, characterized in that: The filtration suction pump (50) is arranged on the downstream side of the filtration part (20) and is used to provide power for filtering the blood sample and collect filtrate; The flushing injection pump (60) is arranged on the downstream side of the filter section (20) to provide power and flushing liquid for reverse flushing of the filter section (20), and / or the flushing injection pump (60) is arranged on the upstream side of the storage section (10) to provide power and flushing liquid for forward flushing of the storage section (10).

7. A capture method of the high-throughput fully-automatic cell separation and capture device according to any one of claims 1 to 6, characterized in that: include: a preloading step, loading a red blood cell lysing solution into the pipeline between the first three-way valve (70) and the storage part (10) through the lysing solution injection part (30), and loading a blood sample to be filtered into the storage part (10); A control parameter setting step, inputting the control parameters of the initial sample volume, the filtration suction pump (50) and the flushing injection pump (60) through the user interface of the control program, wherein the control parameters at least include a flow rate parameter and a flow rate parameter; an automatic filtering step, wherein the blood sample is filtered through a filter unit (20) under the control of a controller (100), and target cells are obtained in the filter unit (20), wherein after the blood sample passing through the filter unit (20) reaches a first set amount, the filter unit (20) is flushed with a second set amount of flushing liquid, and the first set amount is greater than the second set amount; an automatic lysis step, under the control of the controller (100), the flushing injection pump (60) drives the red blood cell lysis solution into the storage portion (10), and lyses the red blood cells retained in the storage portion (10) for a set time; an automatic flushing step, during the filtering process, after the blood sample passing through the filter part (20) reaches a first set amount, the filter part (20) is backwashed with a second set amount of flushing liquid to prevent the microporous screen (22) from being blocked, wherein the first set amount is greater than the second set amount. After the red blood cell lysis is completed, the flushing injection pump (60) and the filtration suction pump (50) will work alternately to complete the washing of the enriched cells; The suspension step includes introducing gas from the downstream side of the filter section (20) so that the target cells are separated from the microporous sieve (22) in the filter section (20), suspended in the air cushion liquid phase of the storage section (10), and recovered by a pipette.

8. The capture method of the high-throughput fully-automatic cell separation and capture device according to claim 7, characterized in that: Before the suspension step, the method further comprises: The storage part (10) is flushed a set number of times after the red blood cells are lysed. During the flushing process, the filter part (20) is flushed from the downstream side of the filter part (20) with a second set amount of flushing liquid and enters the interior of the storage part (10), and the storage part (10) is flushed from the upstream side of the storage part (10) with a third set amount of flushing liquid, wherein the third set amount is between the second set amount and the first set amount.

9. A high-throughput cell separation and capture system, characterized in that: include: A controller (100) is used to set, control and display the motion parameters and motion states of the filtration suction pump (50), the flushing injection pump (60) and the electromagnetic three-way valve (90), thereby controlling the motion and direction of the fluid in the system to complete the filtration of the sample and the washing of the enriched cells; The high-throughput fully-automatic cell separation and capture device as described in any one of claims 1 to 6 above, or, when separating cells, a capture method using the high-throughput fully-automatic cell separation and capture device as described in claim 7 or 8 above; The controller (100) is connected to the filtering suction pump (50), the flushing injection pump (60) and the electromagnetic three-way valve (90) and is used to automatically filter, lyse and flush the blood sample in the storage part (10) to obtain high-purity target cells.

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