Centralized cell sorting system and sorting method

[0077] Example one

[0078] The present embodiment provides a centralized cell sorting system, such as Figure 1 - Figure 4 , The sample cell suspension comprising the target cells containing the 250 target cells were enriched microfluidic 250 cell sorting apparatus 120, and the driving into the sample cell suspension microfluidic cell sorting 120 of the first driving means is selected from mechanism; microfluidic cell sorting device is provided with a sample supply conduit A150 B160 waste conduit 230 for the outflow and the outflow of the target cells 250 120; also be provided separate from the filter pipe B160 of target cells on the micro-pipe 250 B160 pore membrane device 110.

[0079] Specifically, the first drive mechanism may be a peristaltic pump, a syringe pump, a diaphragm pump, a gear pump, piston pump, an electroosmotic flow pump or the like for driving the cell suspension into the microfluidic cell 120 of the prior art sorting apparatus.

[0080] Potential to cause filter clogging through microfluidic cell sorting means for removing a sample of cell suspension majority of particles such as non-target cells ingredients, reduce and prevent cell suspension sample flows through the microporous membrane means in the microfluidic device cell sorting action, target cells and a sample waste separation, cell sorting achieve further filtered through a microporous membrane means, the target cells were concentrated in the microporous membrane means, to improve the cell sorting efficiency.

[0081] In particular, the separation system for enriching a sample from the number of rare cells with a large amount of background target cells, and recovery of the sample solution was concentrated, the target cells are concentrated in a small range, to facilitate subsequent processing . like figure 1 , The main components within the dashed box means is a sample pretreatment system 100, the sample pretreatment system comprising: a target cell 250 for enriched microfluidic cell sorting apparatus 120, the sample injection line 130, buffer conduit 140, conduit A150, B160 pipe, the buffer container 180 and waste container 190. Sample container to be treated outside the broken line 170, the recovery liquid was filtered and microporous membrane means container 200 for recovering the concentrated solution 110; recovered liquid after the recovered liquid tank 200 was collected by filtration 240; 240 of the main liquid recovered a buffer and micro cells.

[0082] Wherein when the first drive means is a syringe pump, the syringe pump injection rate is controlled in the range of 0.05 ~ 50mL / min, wherein, if the first drive mechanism is a peristaltic pump, a pump of the peristaltic pump tube diameter is 0.25 to 2 mm, the flow control in the range of 0.05 ~ 50mL / min, all other lines are narrow tube inner diameter of 0.25 to 2 mm.

[0083] Specifically, microfluidic cell sorting apparatus 120 is a passive sorting spiral flow path inertial focusing microfluidic chip, laterally offset uncertainty microfluidic chips, the microfluidic chip micro vortex or tangential flow filtration micro fluidic chip.

[0084] Specifically, microfluidic cell sorting means 120 may also be using an electric field, magnetic field, ultrasound, optical tweezers, etc. Active sorting microfluidic cell sorting apparatus.

[0085] In particular, the microporous membrane comprises a microporous membrane means 110 (not shown) and a microporous membrane (not shown) is fixed an annular member (not shown) at the output terminal B160 pipe; annular member (not shown) tightly sleeved B160 at the output of the pipeline;

[0086] Specifically, the aperture diameter of the microporous membrane is less than 250 target cells; wherein the typical range for microporous membrane pore size is 3 to 15 m;

[0087] Specifically, the porosity of the microporous membrane is not less than 20%, in particular, the pores of the porous membrane was 80%;

[0088] In particular, the microporous membrane is not larger than 4㎜²;

[0089] Specifically, microporous membrane, wherein the microporous membrane means are arranged in a circular diameter 1.5 mm, thickness 15 m, pore size 7 m, the rules;

[0090] It is noted that, for ease of description the method of the present invention, a key step, other components of the sample preparation system 100 figure 1 It is omitted, but in the actual device design and manufacturing process, the sample should also include a buffer and a flow control unit and the input needed, process control systems, human interface device housing and corresponding monitoring and protection devices.

[0091] figure 2 Further describes a solution comprising a portion of said second fluid control means symbology. Wherein the reagent switch valve 270, the temperature control system 280, temperature sensor 290, the reagent injection means 300, a reagent switching means 310, 320 the reagent, the reagent container 330, the reagent injection line 340, check valve 350, together constitute a second means like labeled cells automated operation. The second driving means further comprises a reagent injection fluid in the line enters the second drive mechanism 110 a microporous membrane means, the second drive mechanism is a peristaltic pump, a syringe pump, a diaphragm pump, a gear pump, piston, power prior art flow pumps.

[0092] exist figure 2 A plurality of reagent containers 330 shown, with the target cell 110 for the microporous membrane processing apparatus 320 reagent. When the sample pretreatment system 100 after completion of sample cells were sorted, the target cells to collect all the microporous membrane device 110, the second means if necessary, under the control of a reagent switching device 310, through the reagent feed the sampling device 300 are respectively 320 different reagents were added to the microporous membrane device 110, and complete the fixing of the microporous membrane device 110 with the cells in the temperature control system 280 and temperature sensor 290, closed, playing hole, dyeing, cleaning and other treatment. Microporous membrane 110 is removed after completion of the step from the pipe B160, identified for analysis under a microscope, or cells within the porous membrane in the device 110 to other analytical instrumentation analysis. Due to the small area of ​​the microporous membrane filter device 110 can be viewed under a microscope field of all the cells, so cells were analyzed rapidly, without having to configure the microscope mobile internet.

[0093] It is noted that, for ease of description of the present invention, a key step in the method, part of the device in the second member figure 2 It is omitted, but in the actual device design and manufacturing process, the flow control unit also comprising reagents needed, process control systems, human interface device housing and corresponding monitoring and protection devices.

[0094] It should be pointed out that figure 2 Said second means only in the microporous membrane of the cell device 110 a program example of processing, in the specific implementation process, the type, quantity and injection mode agent will need to be determined and adjusted according to the protocol. Wherein the nature of the reagents may be two, three, four, five, six, seven, eight, nine, ten.

[0095] image 3 for figure 2 A more specific embodiment of the system shown, to illustrate different types of components when a functional unit, a system for possible presentation. image 3 Microfluidic cell sorting apparatus 120 shown laterally offset deterministic microfluidic chip; laterally offset uncertainty microfluidic chip includes a laterally offset region of uncertainty; uncertainty region laterally offset A side comprising an inlet, an outlet on the other side B comprising; A comprise at least two inlet channels, one channel for sample input, another input channel for buffers; B comprising at least two outlet channels; channel one for the target cell and the buffer flow, waste effluent samples for the other channel; in one particular application, the effluent waste sample lateral offset uncertainty region was recovered by filtration and flow ratio can be 1: 1,2: 1,3 : 1,4: 1,5: 1,6: 1 and the like;

[0096] Lateral offset uncertainty region comprises a plurality arranged in an array and the flow of the sample at an oblique angle of the guide post (not shown); In one particular application, adjacent guide post (not shown) between the spacing may be 50μm ± 5μm; 250 target cells in contact with the guide post made (not shown) in a predetermined direction offset trace, the target cell 250 with the other ingredients of the sample separated and discharged from the duct with buffer B160, and 230 samples from the remaining waste liquid discharge pipe A150;

[0097]Specifically, the sorting system using an injection pump driving manner to be treated sample container sample to be processed within the 170 210 suction syringe pumps according to a set flow rate through the sample and the sample transport line 130 to the microfluidic cell sorting 120. Wherein the sample cell suspension into a syringe pump to a microfluidic cell sorting apparatus 120 of the switching valve 440 is controlled by the second switch. At the same time, the sorting system using an injection pump driven manner in the buffer, the buffer container 180220 suction syringe pumps 140 and delivered to the microfluidic cell sorting apparatus 120 according to a set flow rate through the line buffer to ensure that the sample and buffer into the microfluidic cell sorting according to a certain proportion of the apparatus 120, in order to achieve sorting of cells enriched. Wherein the buffer solution into the syringe pump to the microfluidic cell sorting switching device 120 is controlled by the third switching valve 450. By cell sorting microfluidic device 120 for recovering the treated fluid into the porous membrane 110 through line B160. Sample processing is complete, all of the target cell 250 in the microporous membrane device 110 enriched in.

[0098] When the target cell 250 enrichment, a sample containing the target cells to be treated 250 by the sample injector 210 flows into conduit 130 cell sorting microfluidic device 120, under the action of the buffer line 140 through the buffer 220 is introduced, 250 target cells in a microfluidic cell sorting apparatus 120 with the other components separated from the sample, sample waste 230 into the waste container 190, the target cell 250 via outflow conduit B160 enriched effluent sample preparation system 100 through the conduit A150, apparatus 110 into the microporous membrane, the membrane pore size microporous membrane since the device 110 is less than the diameter of the target cell 250, target cell 250 will thus be trapped within the microporous membrane device 110, and other liquid recovered following the target cell 250 B160 than the inflow conduit of the solids component of a target cell 250 of a small sample liquid flows into the recovery vessel 200 through hole 110 microporous membrane filter means.

[0099] When all 210 samples are to be processed through the cell sorter microfluidic separation device 120, the entire sample to be processed 210 the target cell 250 is trapped in the porous membrane 110 above the filter means. A second switching means 360 reagent valve 270 to the flow path switching B160 conduit communicates with the second device 360.

[0100] Specifically, the second means 360 further comprises a reagent switching valve 270, the temperature control system 280, the reagent injection means 300, a reagent switching means 310, an air filtering device 430 and 320 by the reagent, the reagent containers 330, 340 constituting the reagent injection line five reagent loading unit and the like. Five reagent loading unit, the air filtering apparatus 430, the reagent injection means 300 are connected to a reagent switching means 310; wherein the air filtering device 430 filtration accuracy of 0.2 m; device 300 may be a peristaltic pump, the peristaltic pump tubing reagent injectors an inner diameter of 0.5 mm, the flow rate control range of 10 to 100 microliters per minute.

[0101] like image 3 Switching, when the switch valve 270 to the reagent flow path pipe B160 communicates with the second switching device 360, the reagent injection device 300 according to the five kinds of reagent 320 by the reagent are sequentially programming the switching means 310 are sequentially loaded into the microporous membrane device 110, thereby fixing the microporous membrane to achieve device 110 cells, permeabilized, blocked, dyeing, cleaning and other treatment. When the load some reagents necessary to control the temperature of the microporous membrane device 110, 280 by the temperature control pipe by controlling the temperature of B160, and varying the temperature of the microporous membrane device 110 through heat conduction. When the microporous membrane device 110 requires a reagent emptying, switching means 310 to the reagent reagent injection apparatus 300 and the effect of an air filtering device 430 is turned on, and a peristaltic pump, in the porous membrane 110 the reagents were drained. May be removed through the second means 360 treated microporous membrane device 110 from the sample by way of conduit B160 microporous membrane separating device 110, identified and analyzed under a microscope or other analytical device microporous cells within the membrane device 110 for analysis. Due to the small area of ​​the microporous membrane filter device 110 can be viewed under a microscope field of all the cells, so cells were analyzed rapidly, without having to configure the microscope mobile internet. In carrying out the process cell, avoiding the need for conventional treatment frequently centrifugal cleaning step, cleaning time saving, but also to minimize the loss and damage of cells.

[0102] For cells were PCR (polymerase chain reaction analysis) or NGS (high-throughput sequencing), the residue from the bottom of the apparatus 110 within the microporous membrane of a cell lysate through a filter paper after the cells were lysed absorbance. The same operation described above avoids the need for the volume of the concentrated cells were centrifuged step.

[0103] It is noted that, for ease of description of the present invention, a key step in the method, the cell concentration in the sorting system design and manufacturing processes in an actual apparatus, it may further comprise a sample and necessary buffers and flow control means input, process control systems, human computer interface device housing and corresponding monitoring and protection devices.

[0104] One particular use may be, the de human peripheral blood circulating tumor cells were enriched by immunofluorescence staining and automated. For example, first need to sample preparation system 100 precharge buffer, comprising the step of addition of 7.5 ml human peripheral blood samples to be processed within the vessel 170, 50 ml of 1 × PBS buffer in a buffer reservoir 180, the third the switching valve 450 is switched to the buffer agent injection pump 420 and conduit 140 communicating state buffer, at a flow rate of 30 ml per minute buffer reagent into the syringe pump sample buffer 420; and the second switching valve 440 is switched the pump 260 to the feeding line 130 and the sample injector communicating state while the third switching valve 140 communicates the status to the buffer agent injection pump 420 and line buffer 450 is switched, at a flow rate of 500 microliters per minute buffer liquid conduit 140 is filled by the buffer microfluidic cell sorting apparatus 120, and held for one minute. Buffer reagent injection pump 420 to the line buffer system during the precharge period, the first injection pump 260 at a flow rate of 100 microliters per minute suction portion of the buffer, then the second switching valve 440 is switched to the injection pump 260 samples to be processed with the communication status container 170, at a flow rate of 100 microliters per minute discharge portion buffer, the sample pretreatment system and the gas discharge conduit for all the light in the syringe 100, the precharge is completed. Next, the sample needs to be loaded into the injection syringe pump 260, including the step of at a flow rate of 10 ml per minute inhalation entire sample solution, while ensuring that the gas is not sucked into the syringe, the injection pump 260 is stopped and the buffer agent injection pump 420, and the sample passage switching the second switching valve 440 is switched to the injection pump 260 and the sample injection line 130 communicates status. May begin after the end of the sample prepared above process procedure, the process comprising, at the same time opening the injection pump 260 and a buffer agent injection pump 420, respectively, at a flow rate of 0.2 ml per minute with 0.5 ml cell suspension per minute and samples injection buffer cell sorting microfluidic device 120, under the action of laterally offset uncertainty, the cell suspension in the sample isolated circulating cells from the tumor, and under the action of the circulating apparatus 110 of the microporous membrane tumor cells in the interception means 110 microporous membrane. When the suspension after treatment of the cells within the sample injection pump 260 first stops the pump 260 flows into the sample, reagent and buffer injection pump 420 continuously pumps the buffer for about 1 minute, the microfluidic cell sorting device after all of the residue with 120 discharge conduit A150 cell suspension in the sample buffer to stop the reagent injection pump 420, the sorting is completed.

[0105] Specifically, five reagent container 330 are respectively provided with 1 × PBS buffer, fixing solution of 4% paraformaldehyde, permeabilized agent 0.4% Triton X-100, a blocking solution and DAPI / CD45 / CK staining reagent cocktail. When the switching valve 270 to the reagent flow path pipe B160 communicates with the second switching device 360, a reagent switching means 310 is switched to the first state of the communication pipe 330 and the reagent container B160 with 1 × PBS buffer, the reagent injection means 300 1 × PBS buffer at a flow rate of 0.1 ml per minute to load the microporous membrane device 110 for washing is stopped 2 minutes after loading, then the switching device 310 is switched to the reagent conduit B160 with fixative containing 4% formaldehyde reagent container 330 communicating state, by a peristaltic pump in a fixative solution of 0.1 ml per minute flow rate is loaded into the microporous membrane device 110 for loading loading stopped after 10 seconds, and for ten minutes, sufficient to allow the cells fixed , prevent proteolytic enzymes induced autolysis and cell decay, enhanced hardness and mechanical strength of the cells, after a reagent switching means 310 is switched again to the state of the communication pipe 330 and the reagent container B160 with 1 × PBS buffer, by a peristaltic pump after 1 × PBS buffer was loaded at a flow rate of 0.1 ml per minute to a microporous membrane device 110 for cleaning for 2 minutes, washed excess fixative stop loading, after a reagent switching means is switched to the conduit 310 and with B160 permeabilizing reagent container 0.4% Triton X-100 in the communication state 330, the permeabilizing agent at a flow rate of 0.1 ml per minute by a peristaltic pump to the loading device 110 microporous membrane for 10 seconds after loading the loading stops, and for ten minutes to rupture the cells were permeabilized, passage holes to play or the antibody on the cell membrane and in contact with the dye readily enter the intracellular protein, the reagent after the switching means 310 switches again to a conduit equipped with 1 × PBS B160 buffer reagent container 330 communicating state, by a peristaltic pump 1 × PBS buffer at a flow rate of 0.1 ml per minute to load the microporous membrane device 110 for cleaning for 2 minutes, stopped after loading cleansed excess permeabilizing agent after reagent communication state switching means 330 is switched to the conduit 310 and the reagent container containing B160 blocking solution, the blocking solution at a flow rate of 0.1 ml per minute to load the microporous membrane device 110 by a peristaltic pump for 10 seconds loading after stopping loading, and held for thirty minutes in order to reduce the subsequent dyeing process, or non-specifically bound antibodies and the sample dye, then switching means 310 switches again reagent to the reagent container containing B160 duct 1 × PBS buffer status communication 330 , the peristaltic pump 1 × PBS buffer at a flow rate of 0.1 ml per minute to load the microporous membrane device 110 for cleaning for 2 minutes, stopped after loading the excess blocking solution was washed off, after the switching device 310 is switched to the reagent conduit B160 is equipped with a fluorescent staining reagent cocktail reagent container 330 communicating state, by a peristaltic pump at a flow rate cocktail staining reagent 0.1 ml per minute to a microporous membrane loading device 110 for loading after 10 seconds to stop loading, and holding sixty minutes, for fluorescent microporous membrane device 110 cells Light staining staining process, after the switching means 310 switches the reagent 330 to the communication state again and the reagent container containing conduit B160 1 × PBS buffer, the peristaltic pump 1 × PBS buffer at a flow rate of 0.1 ml per minute to load the microporous membrane device 110 for cleaning for 2 minutes, washed cocktail after the excess staining reagents stop loading, from the completion of the fixed microporous membrane device 110 cells, permeabilized, blocked and staining process.

[0106] Circulating tumor cells processed by the microporous membrane sample can be analyzed to identify the device 110 separating the sample pretreatment system 100, and placed under the inverted fluorescence microscope.

[0107] This application example of a job by avoiding frequent washing step centrifugation, automated staining process flow of the significantly shorter time required for the process, but also reduces the loss of cells caused by the centrifugal cleaning process. On the other hand, due to the small area of ​​the porous membrane filtration membrane device 110, to facilitate the cell membrane navigate inverted fluorescence microscope, and was observed to find where the circulating tumor cells. Means the presence of a microporous membrane 110 may also be a small amount of blood cells into the conduit B160 in the sample preparation system 100,420 non expected traffic fluctuation occurs in the injection pump 260 and the buffer agent injection pump, these blood cells filtered out of the microfluidic pores.

[0108] Example 2

[0109] The present embodiment provides a centralized cell sorting system, such as Figure 4 Distance Figure 4 for figure 2A more specific embodiment of the system shown embodiment, when employed for describing different types of functional unit means, another possible presentation system. And a pretreatment system 100 comprises a second device 360, wherein the microfluidic cell sorting apparatus 120 may further focus the spiral flow path inertial microfluidic chip; spiral flow path inertial focusing microfluidic chip includes a plurality of turns helically disposed as a stream channel; end of the flow channel comprises an inlet C for the suspension of cells was diluted sample input, the other end comprising an outlet D; D comprises at least two outlet channels, one channel for the outflow of target cells and buffers, samples for the other channel waste effluent; in one particular application, the waste effluent sample after the spiral flow path and recovery flow filtration ratio may be 1: 1,2: 1,3: 1,4: 1,5: 1,6: 1 and so on;

[0110] The radius of curvature of the spiral flow path 5 to 9㎜, trapezoidal cross section, 80 m deep inside, outside 130 m deep, wide 600μm.

[0111] Microporous membrane 110 is a microporous membrane means circular diameter 1.5 mm, thickness 15 m, 7 m pore size, regularly arranged, a porosity of 80%.

[0112] Inertial spiral flow path for focusing microfluidic sample cell suspension shunt. The sorting system driven by constant voltage manner of suspension of cells in the sample sample preparation system 100 through the sample 130 to the sample delivery line spiral flow path inertial focusing microfluidic chip. Wherein the delivery and the start control 370 is turned off by the injection valve. Be treated sample container 170 by the pressure in the injection pump regulator 260 and voltage regulator 380, the pressure regulator means 380 of the controller; the sample into sample flow line 130 and the spiral flow path inertial focusing microfluidic chip in line with sorting requirements.

[0113] Figure 4 In the sample to be processed side of the container 170 is also connected to means for removing solid particles to be filtered in the process gas in the sample container 170 390. Filtration accuracy of the filter means 390 is 0.2μm.

[0114] By cell sorting microfluidic device 120 for recovering the treated fluid into the porous membrane 110 through line B160. After the completion of the sample processing of target cells in the entire microporous membrane enrichment apparatus 110, the agent 360 of the second switching valve 270 means the flow path switching B160 conduit communicates with the second device 360.

[0115] Cell sorting microfluidic device 120 may also be a micro vortex microfluidic cell sorting or cross-flow filtration apparatus (also referred to as tangential flow filtration) prior art chips.

[0116] Specifically, such as Figure 4 , The second valve means 360 is switched from the agent 270, the temperature control system 280, 320 and the reagent, the reagent container 330, the reagent injection line 340, and a reagent syringe pump 400 switches the first valve 410 is configured to load three reagent unit or the like. The first switching valve is mainly used for injection of a reagent syringe pump cells were treated with supplemental injections switching state; wherein the injection capacity of the injection pump 400 as a reagent 1mL, flow control range of 10 ~ 1000μL / min All lines are 0.5 mm inner diameter PEEK tubing.

[0117] When the switching valve 270 to conduit reagent B160 is switched to the flow path and the second communication device 360, respectively, through three reagent reagent loading unit 340 and the reagent injection line injection pump 400 ON state of the first switching valve 410 is switched to, 320 reagent from the reagent container 330 by the reagent injection line 340 added to the reagent injection syringe pump 400, after which the first switching valve 410 is switched to the ON state of a pipe 400 with a reagent injection pump B160, whereby the reagent syringe pump 400 reagent loading device 110 to the microporous membrane, thereby achieving fixing of the microporous membrane device 110 cells, permeabilized, blocked, dyeing, cleaning and other treatment. When the load some reagents necessary to control the temperature of the microporous membrane device 110, 280 by the temperature control pipe by controlling the temperature of B160, and varying the temperature of the microporous membrane device 110 through heat conduction. May be removed through the second means 360 treated microporous membrane device 110 from the sample by way of conduit B160 microporous membrane separating device 110, identified and analyzed under a microscope or other analytical device microporous cells within the membrane device 110 for analysis. Due to the small area of ​​the microporous membrane filter device 110 can be viewed under a microscope all cells, so cells were analyzed rapidly, without having to configure the microscope mobile internet.

[0118] like Figure 4 Shown, another specific use of the second means may be, human peripheral blood to remove red cells in circulating tumor cells were enriched and automated pretreatment immunofluorescence staining. For example, the addition of the sample container 170 to be treated 5 ml 1 × PBS buffer and the vessel was sealed, closing the injection valve 370, opening the injection pump 260, and under the action of the regulator device 380 will be processed within the sample container 170 the pressure was adjusted to 0.1 MPa. Opening the injection valve 370 and held for 2 minutes to microfluidic cell sorting apparatus 120 and conduit pipes A150 and B160 injection valve 370 after closing the entire buffer is full. After 7.5 ml of human peripheral blood erythrocyte lysis, with 1 × PBS buffer, resuspended to 15 ml, transferred to a sample to be processed within the vessel 170 to be treated and the sample container 170 closed. Injection pump 260 is turned on again, and under the action of the regulator device 380 to be treated sample vessel 170 was adjusted to 0.085 MPa pressure, the sample cell suspension at a flow rate of 1.7 milliliters per minute through the entire microfluidic cell sorting 120. Microfluidic cell sorting device 120 and the focusing action of inertia Dean vortex effect, when the sample cell suspension to flow to the end of the spiral flow path, circulating tumor cells will gather at a shallow flow passage inside and through the recovery B160 inlet duct flow passage, a microporous membrane wherein the intercepting means 110 cells, and the liquid ingredients into the recovery liquid tank 200; and white blood cells in the sample while remaining outside the erythrocyte aggregation deeper flow channel and through the waste liquid passage into the conduit A150, waste container 190 is ultimately collected.

[0119] Specifically, three reagent containers 330, respectively, with 1 × PBS buffer, fixing solution of 4% paraformaldehyde, and from 5% goat serum, 5% FcR blocking reagent, 0.4% Triton X-100 and 0.3M glycine 1 × PBS permeabilization mixed solution composed of - a closed premix fluid. When the switching valve 270 to the reagent flow path pipe B160 is switched to the second communication device 360 ​​after the first three reagent loading unit through the first switching valve 410 is switched to the reagent and the reagent injection line 340 connected to the syringe pump 400 state, the reagent from the reagent container 330 through 320 added to the reagent injection syringe pump reagent injection line of 340,400, followed by the first switching valve 410 is switched to the conduit and a reagent syringe pump B160 with 1 × PBS buffer contact state, the agent from the syringe pump 1 × PBS buffer at a flow rate of 0.1 ml per minute to load the microporous membrane device 110 for washing is stopped 2 minutes after loading, then the first switching valve 410 is switched to the conduit B160 with 4% paraformaldehyde fixative syringe pump ON state, the fixing solution at a flow rate of 0.1 ml per minute to a microporous membrane loaded by the agent apparatus 110 syringe pump for 10 seconds after loading the loading stops, and for ten minutes, sufficient to allow the cells is fixed, then again 1 × PBS buffer at a flow rate of 0.1 ml per minute to load device 110 by a microporous membrane reagent syringe pump for 2 minutes to stop loading washing, after the first a switching valve 410 is switched to the conduit and with permeabilization B160 - blocking agent premix liquid injection pump on state, the agent from the syringe pump permeabilized - blocking a flow rate of premix was loaded onto a 0.1 ml per minute microporous membrane means 110 for loading stops after 10 seconds to load, and held for thirty minutes to carry out blocking treatment rupture permeabilized cells and, after 1 × PBS buffer injection pump to the reagent flow rate 0.1 ml per minute again microporous membrane 110 is loaded into the apparatus for cleaning for 2 minutes after stopping the loading of the microporous membrane from the completion of the fixed device 110 cells, permeabilized blocking treatment.

[0120] The processed samples may be circulating tumor cells by the microporous membrane separation device 110 from the sample pretreatment system 100, with further immersed in 96 well plates cocktail fluorochrome for 1 hour, and by 1 × PBS buffer Soak several times to remove excess stain to complete the staining of the sample. Stain finish sample of cells can be analyzed and identified under an inverted fluorescence microscope. As a result of staining takes a long time, the microporous membrane device 110 detached independently dyeing process can save a lot of time to minimize the manual steps by way of a plurality of samples simultaneously in parallel.

[0121] This application example of a job by avoiding frequent washing step centrifugation, automated staining process flow of the significantly shorter time required for the process, but also reduces the loss of cells caused by the centrifugal cleaning process. On the other hand, due to the small area of ​​the porous membrane filtration membrane device 110, to facilitate the cell membrane navigate inverted fluorescence microscope, and was observed to find where the circulating tumor cells.

[0122] In this embodiment, the sample pretreatment system 100 for sorting enriched sample used time within ten minutes, and the second enrichment device 360 ​​to the target cells were fixed, permeabilized, although the washing process than the use of a closed centrifuge program to save time, a sample handling process is still more than 45 minutes. When needed to process more samples, sample preparation system 100 can be operated independently and run with the second separating apparatus 360 through.

[0123] like Figure 5b As shown, the second device 360 ​​may be further provided with a plurality of reagent reagent dispensing outlet line control valve 480, control the outflow of the reagent 470, 460 and a reagent reagent dispensing line 490. The waste liquid collecting container; reagent waste collection container 490 flowing the reagent accommodated in the outlet line 480 from the waste liquid 500. Each reagent outlet 360 of the second conduit means 480 can be a socket, in which the cells were stained for the correlation process 100 is separated from the apparatus 110 with a microporous membrane sample cell sample pretreatment system.

[0124] Specific methods include such Figure 5A Shown with the second device 100 without sample pretreatment system 360, a sample sorting enriched enriched microporous membrane after completion of separation apparatus 110 and the sleeve 100 from the sample preparation system such as to Figure 5b A second independent means 360 shown in FIG. When the second sample requires more concurrent processing apparatus 360 can simultaneously a plurality of sockets 110 on a microporous membrane means independent of the second means 360, controlled by the control valve 470 reagent dispensing apparatus 110 in the microporous membrane the cells were loaded reagents. Loading process may be carried out simultaneously in all the sets of the microporous membrane device 110 to the second device 360, i.e., while the reagent dispensing control valve 470 is opened, to the microporous membrane device 110 cells were loaded with reagents, and also may timeshare, i.e., after the first sorting process is completed through the sample pretreatment sample pretreatment system 100, a microfiltration membrane separation apparatus 110 to the first sleeve and a second reagent outlet conduit means 360 480, and starts before the dyeing process, this time with other agents reagent dispensing outlet line 480 connected to the control valve 470 is in the full closed state. When the second sample through the sample pretreatment system 100 pre-sorting process is completed, the porous membrane separating means 110 to the second set of reagent and an outlet conduit 480 of the second device 360, and the first when samples held in a state where the reagent is loaded, the control valve 470 is opened and the reagent dispensing a second reagent outlet conduit 480 is connected to, and cleaning agents, such as load operations, and so forth three to three and situation above the sample. Since the holding state is significantly longer than the time required for loading and wash buffer reagent, it is easy to realize a plurality of time-sequential processing of samples.

[0125] Example three

[0126] The present invention also includes a cell sorting method, applying the concentrated cell sorting system; such as Image 6 Shown, comprising the steps of:

[0127]Step 1: The cell suspension sample containing target cells is introduced into the microfluidic cell sorting apparatus via the sample induction cytometry, and the main non-target particle ingredients are removed by microfluidic cell sorting means;

[0128] Step 2: Introducing the sorting cell suspension sample in step one, in the fixed flow channel into the microporous filter membrane, the microporous filter device filter is filtered out of partially non-target particle ingredients and waste liquid, whileThe target cells complete selection within the microporous filter device;

[0129] Step 3: Separate the microporous filter device in step II containing the target cells from the fixed flow path.