CN113846012A

CN113846012A - Cell centralized sorting system and sorting method

Info

Publication number: CN113846012A
Application number: CN202111427643.0A
Authority: CN
Inventors: 关国峰; 钞书哲
Original assignee: Shenzhen Genliu Technology Co ltd
Current assignee: Shenzhen Genliu Technology Co ltd
Priority date: 2021-11-29
Filing date: 2021-11-29
Publication date: 2021-12-28

Abstract

The invention relates to a cell centralized sorting system and a sorting method, which comprises a microfluidic cell sorting device for enriching target cells of a cell suspension sample containing the target cells, and a first driving mechanism for driving the cell suspension sample to enter the microfluidic cell sorting device; the microfluidic cell sorting device is provided with a pipeline A for the sample waste liquid to flow out and a pipeline B for the target cells to flow out; the pipeline B is also provided with a microporous filter membrane device for filtering target cells; the microfluidic cell sorting device is used for removing most of particle components such as cells in a sample, reducing and avoiding the possibility of filter membrane blockage caused when the sample flows through the microporous filter membrane device, separating target cells from sample waste liquid under the action of the microfluidic cell sorting device, realizing cell sorting, further filtering through the microporous filter membrane device, concentrating the target cells in the microporous filter membrane device, and improving the cell sorting efficiency.

Description

Cell centralized sorting system and sorting method

Technical Field

The invention relates to the technical field of cell sorting, in particular to a centralized cell sorting system and a centralized cell sorting method.

Background

In a liquid biopsy based on cells, abnormal cells such as circulating tumor cells can be enriched from blood by size and rigidity of cells and cell clusters. In the sorting and enriching method based on physical characteristics, the sorting and enriching method can be roughly divided into two types of sorting and enriching based on a microfluidic chip and sorting and enriching based on a microporous membrane.

Because of the huge number of cells in blood, great pressure is brought to sorting and enrichment. In the sorting and enriching technology based on the microporous membrane, the conventional microporous membrane has low porosity and is difficult to support blood filtration of more than 5 mL. In order to improve the porosity and ensure the uniformity of the diameter of the micropores, only a filter membrane with regularly arranged micropores can be designed and manufactured, and although the flux problem can be solved, the production cost is too high, and the filter membrane is difficult to accept by patients as a medical detection consumable. Sorting and enrichment by filtration generally requires washing the enriched target cells for analytical identification. On-chip identification has two problems, one is that the presence of a filter membrane will cause inevitable interference with optical analysis; another is that the area of the filter membrane is usually large in order to increase the sample flux, so when analyzing with a microscope or other instrument, the whole area needs to be scanned in a full coverage manner to avoid omission, which leads to significant increase of the analysis time and cost.

The cell sorting by the microfluidic technology mainly comprises an inertial focusing microfluidic technology, a deterministic directional offset technology and the like, and can be carried out by ultrasonic, electric field and other modes under the condition of lower flux requirement. The sorting is carried out by utilizing pure physical methods such as hydrodynamics and the like, so that the problems of blockage and the like caused by filtration can be avoided, and the sorting can be carried out continuously for a long time. In practice, microfluidic sorting also suffers from some technical disadvantages. Such as the need to dilute the sample prior to sorting or the introduction of buffers during sorting, etc., this results in a significant increase in the volume of the recovery fluid. When the number of the recovered target cells is less than one thousand, the recovered sample needs to be concentrated when the subsequent analysis and identification are carried out, and the loss of the precious sample is easily caused during the operation. In some solutions, a method of integrating sorting enrichment and on-chip filtering capture is adopted to fix the enriched cells on a specific area of a chip so as to perform on-chip analysis, but in order to reduce the flow rate during capture and improve the capture success rate, the area of the capture area is usually larger, so that the problems of increased analysis time and cost caused by the need of full-coverage scanning during analysis are also faced.

Disclosure of Invention

The present invention provides a cell centralized sorting system and a sorting method, which are directed to overcome the above-mentioned drawbacks of the prior art.

The technical scheme adopted by the invention for solving the technical problems is as follows: a cell centralized sorting system comprises a microfluidic cell sorting device for performing target cell enrichment on a cell suspension sample containing target cells, and a first driving mechanism for driving the cell suspension sample into the microfluidic cell sorting device; the microfluidic cell sorting device is provided with a pipeline A for the sample waste liquid to flow out and a pipeline B for the target cells to flow out; the pipeline B is also provided with a microporous filter membrane device which can be separated from the pipeline B and used for filtering the target cells.

The cell centralized sorting system can be a spiral flow channel inertial focusing micro-fluidic chip for passive sorting, a deterministic lateral shift micro-fluidic chip, a micro-vortex micro-fluidic chip or a tangential flow filtration micro-fluidic chip.

The cell centralized sorting system can also be a microfluidic cell sorting device which can perform active sorting by utilizing an electric field, a magnetic field, ultrasonic waves, optical tweezers and the like.

According to the cell centralized sorting system, the first driving mechanism can be a peristaltic pump, an injection pump, a diaphragm pump, a gear pump, a plunger pump, an electroosmotic flow pump and the like, and is used for driving cell suspension to enter the prior art of the microfluidic cell sorting device.

The invention relates to a centralized cell sorting system, wherein a microfluidic cell sorting device is a deterministic lateral shift microfluidic chip; the deterministic lateral-offset microfluidic chip comprises a deterministic lateral-offset region; one side of the deterministic lateral offset region contains an inlet a and the other side contains an outlet B; the inlet A comprises at least two channels, one channel is used for inputting the cell suspension sample, and the other channel is used for inputting a buffer solution; the outlet B comprises at least two channels; one channel is used for the target cells and the buffer solution to flow out, and the other channel is used for the sample waste solution to flow out;

the invention relates to a cell centralized sorting system, wherein a microfluidic cell sorting device is a spiral flow channel inertial focusing microfluidic chip; the spiral flow channel inertial focusing micro-fluidic chip comprises a plurality of circles of flow channels which are spirally arranged; one end of the flow channel comprises an inlet C for inputting the diluted cell suspension sample, and the other end of the flow channel comprises an outlet D; the outlet D comprises at least two channels, one channel is used for the target cells to flow out, and the other channel is used for the sample waste liquid to flow out;

the cell centralized sorting system of the invention, wherein the sorting system further comprises a second device for automatically labeling the target cells; the second device is communicated with the microporous filter membrane device;

the cell centralized sorting system comprises a second device, a first driving mechanism and a second driving mechanism, wherein the second device comprises a temperature control system, a reagent switching device, at least one reagent sampling pipeline connected with the reagent switching device and the second driving mechanism for driving fluid in the reagent sampling pipeline to enter the microporous filter membrane device;

the cell centralized sorting system of the invention, wherein the second device further comprises a reagent switching valve for switching the fluid in the second device to flow into the microporous filter membrane device;

the cell centralized sorting system comprises a microporous filter membrane device, a pipeline B and a cell sorting device, wherein the microporous filter membrane device comprises a microporous filter membrane and an annular member for fixing the microporous filter membrane on the pipeline B; the annular member is tightly sleeved on the output end of the pipeline B;

the cell centralized sorting system is characterized in that the pore diameter of the microporous filter membrane is smaller than the diameter of the target cell;

the cell centralized sorting system provided by the invention is characterized in that the porosity of the microporous filter membrane is not less than 20%;

the invention also discloses a cell sorting method, which applies the cell centralized sorting system; the method comprises the following steps:

the method comprises the following steps: introducing a cell suspension sample containing target cells into a microfluidic cell sorting device through a sample introduction pipeline, and removing main non-target particle components in the cell suspension sample through the microfluidic cell sorting device;

step two: introducing the sorted cell suspension sample in the step one into a microporous filter membrane device along a fixed flow channel, filtering out partial non-target particle components and waste liquid by using the microporous filter membrane device, and simultaneously trapping the target cells in the microporous filter membrane device to finish sorting;

step three: separating the microporous filter membrane device which retains the target cells in the step two from the fixed flow channel.

The beneficial effects of the invention are as follows:

(1) the microfluidic cell sorting device is used for removing most of particle components such as cells in a blood sample, reducing and avoiding the possibility of filter membrane blockage caused by the cell suspension sample flowing through the microporous filter membrane device, separating target cells from sample waste liquid under the action of the microfluidic cell sorting device, realizing cell sorting, further filtering through the microporous filter membrane device, concentrating the target cells in the microporous filter membrane device, and improving the cell sorting efficiency. Meanwhile, the area of the filter membrane can be obviously reduced, so that the use cost of the filter membrane and the time of subsequent optical analysis are reduced.

(2) Through the reagent in the second device, target cells are fixed, punched, sealed, dyed, cleaned, cracked and the like, and can be directly carried out on the microporous filter membrane, so that the time waste and cell loss and damage caused by cleaning through centrifugal liquid replacement in conventional operation are avoided.

(3) By concentrating and concentrating the target cells in a smaller area, the operations such as marking the cells can be realized by consuming less reagents in the subsequent analysis, the reagent consumption is effectively reduced, and the analysis cost is reduced.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the present invention will be further described with reference to the accompanying drawings and embodiments, wherein the drawings in the following description are only part of the embodiments of the present invention, and for those skilled in the art, other drawings can be obtained without inventive efforts according to the accompanying drawings:

FIG. 1 is a schematic diagram of the operation of a centralized cell sorting system according to a preferred embodiment of the present invention;

FIG. 2 is a schematic diagram of the main components of the microfiltration membrane device, the sample pretreatment system connected thereto and the second device of the centralized cell sorting system according to the preferred embodiment of the invention;

FIG. 3 is a schematic diagram of the main components of the system for sample pre-processing by deterministic lateral shift microfluidic chip and controlling the reagent in the second device by peristaltic pump in combination with rotary switch valve to process the recovered sample in the microporous filter membrane according to the preferred embodiment of the present invention;

FIG. 4 is a schematic diagram of the main components of a spiral flow channel inertial focusing microfluidic chip for sample pretreatment and the processing of the recovered sample in the microporous filter membrane by controlling the reagents independently in the second device through the syringe pump according to another preferred embodiment of the present invention;

FIG. 5A is a schematic diagram of another system configuration of the pretreatment system of FIG. 4 without the second apparatus;

FIG. 5B is a schematic view of another system main component configuration of the second apparatus of FIG. 4 separated from the pretreatment system;

FIG. 6 is a flowchart of a sorting method according to a preferred embodiment of the present invention.

The reference numbers are as follows:

100 is a sample pretreatment system;

110 is a microporous membrane device;

120 is a microfluidic cell sorting device;

130 is a sample injection pipeline;

140 is a buffer liquid pipeline;

150 is a conduit A;

160 is a pipe B;

170 is a sample container to be processed;

180 is a buffer solution container;

190 is a waste liquid container;

200 is a recovery liquid container;

210 is a sample to be treated;

220 is a buffer solution;

230 is sample waste;

240 is a recovery liquid;

250 is a target cell;

260 is a sample injection pump;

270 is a reagent switching valve;

280 is a temperature control system;

290 is a temperature sensor;

300 is a reagent sample introduction device;

310 is a reagent switching device;

320 is a reagent;

330 is a reagent container;

340 is a reagent sample inlet pipeline;

350 to a check valve;

360 is a second device;

370 is a sample injection valve;

380 is a voltage stabilizer;

390 is a filter device;

400 is a reagent injection pump;

410 is a first switching valve;

420 is a reagent buffer solution sample injection pump;

430 is an air filtering device;

440 is a second switching valve;

450 is a third switching valve;

460 is a reagent dispensing line;

470 is a reagent dispensing control valve;

480 is a reagent outlet pipeline;

490 is a reagent waste liquid recovery container;

500 is waste liquid.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the following will clearly and completely describe the technical solutions in the embodiments of the present invention, and it is obvious that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments of the present invention without inventive step, are within the scope of the present invention.

Example one

This embodiment provides a cell concentration sorting system, as shown in fig. 1-4, comprising a microfluidic cell sorting device 120 for enriching a cell suspension sample containing a target cell 250 with the target cell 250, and a first driving mechanism for driving the cell suspension sample into the microfluidic cell sorting device 120; the microfluidic cell sorting device 120 is provided with a pipeline A150 for the sample waste liquid 230 to flow out and a pipeline B160 for the target cell 250 to flow out; the tube B160 is further provided with a microfiltration membrane device 110 for filtering the target cells 250, which can be separated from the tube B160.

Specifically, the first driving mechanism may be a peristaltic pump, a syringe pump, a diaphragm pump, a gear pump, a plunger pump, an electroosmotic pump, etc. as is known in the art for driving a cell suspension into the microfluidic cell sorting device 120.

The microfluidic cell sorting device is used for removing most of particle components such as non-target cells in a cell suspension sample, reducing and avoiding the possibility of filter membrane blockage caused by the cell suspension sample flowing through the microporous filter membrane device, separating the target cells from sample waste liquid under the action of the microfluidic cell sorting device, realizing cell sorting, further filtering through the microporous filter membrane device, concentrating the target cells in the microporous filter membrane device, and improving the cell sorting efficiency.

Specifically, the sorting system is used for enriching a rare number of target cells from a sample with a large number of background cells and concentrating a recovered sample solution, so that the target cells are concentrated in a smaller range, and the subsequent processing is convenient. As shown in fig. 1, the dashed box is configured as a main component of a sample pretreatment system 100, which includes: a microfluidic cell sorting device 120 for performing enrichment of target cells 250, a sample introduction line 130, a buffer solution line 140, a pipeline A150, a pipeline B160, a buffer solution container 180, and a waste liquid container 190. A sample container 170 to be treated, a filtered recovery solution container 200 and a microfiltration membrane device 110 for concentrating the recovery solution outside the dotted line; collecting the filtered recovery liquid 240 in the recovery liquid container 200; the recovered solution 240 is mainly a buffer solution, a minute cell, or the like.

If the first driving mechanism is an injection pump, the injection flow rate of the injection pump is controlled within the range of 0.05-50 mL/min, if the first driving mechanism is a peristaltic pump, the inner diameter of a pump pipe of the peristaltic pump is 0.25-2 mm, the flow rate is controlled within the range of 0.05-50 mL/min, and all the other pipelines are thin pipes with the inner diameter of 0.25-2 mm.

Specifically, the microfluidic cell sorting device 120 is a passively sorted spiral-channel inertial focusing microfluidic chip, a deterministic lateral-shift microfluidic chip, a micro-vortex microfluidic chip, or a tangential-flow filtration microfluidic chip.

Specifically, the microfluidic cell sorting device 120 may be a microfluidic cell sorting device that performs active sorting using an electric field, a magnetic field, ultrasonic waves, optical tweezers, or the like.

Specifically, the microfiltration membrane device 110 includes a microfiltration membrane (not shown) and an annular member (not shown) for fixing the microfiltration membrane (not shown) to the output end of the conduit B160; an annular member (not shown) fits tightly over the output end of the conduit B160;

specifically, the pore size of the microporous filter membrane is smaller than the diameter of the target cell 250; wherein the typical range of the pore diameter of the microporous filter membrane is 3-15 μm;

specifically, the porosity of the microporous filter membrane is not less than 20%, specifically, the porosity of the microporous filter membrane is 80%;

specifically, the area of the microporous filter membrane is not greater than 4 mm;

specifically, the microfiltration membranes of the microfiltration membrane device are circular with the diameter of 1.5 mm, the thickness of 15 microns and the micropore diameter of 7 microns, and are regularly arranged;

it should be noted that, for the convenience of describing the core steps of the method of the present invention, other components of the sample pretreatment system 100 are omitted in fig. 1, but in the design and manufacturing process of the actual apparatus, sample and buffer input and flow control units, process control systems, human-machine interfaces, apparatus housings and corresponding monitoring and protection devices, etc. should be included as required.

Fig. 2 further illustrates a solution comprising said second device part by means of a fluid control symbol system. The reagent switching valve 270, the temperature control system 280, the temperature sensor 290, the reagent sampling device 300, the reagent switching device 310, the reagent 320, the reagent container 330, the reagent sampling pipeline 340, and the check valve 350 together form a second device for performing operations such as automatic labeling on cells. The second device further comprises a second driving mechanism for driving the fluid in the reagent sampling pipeline to enter the microfiltration membrane device 110, wherein the second driving mechanism is a peristaltic pump, an injection pump, a diaphragm pump, a gear pump, a plunger pump, an electroosmosis flow pump and other existing technologies.

In the plurality of reagent containers 330 shown in FIG. 2, reagents 320 for treating target cells in the microfiltration membrane device 110 are contained. When the sample preprocessing system 100 finishes sorting the cells in the sample and collects all the target cells into the microfiltration membrane device 110, the second device sequentially adds different reagents 320 into the microfiltration membrane device 110 through the reagent sampling device 300 under the control of the reagent switching device 310 according to the requirement, and finishes the treatments of fixing, sealing, perforating, staining, cleaning and the like of the cells in the microfiltration membrane device 110 under the cooperation of the temperature control system 280 and the temperature sensor 290. After the step is completed, the microfiltration membrane device 110 is removed from the channel B160 for analysis under a microscope or analysis of the cells in the microfiltration membrane device 110 by other analysis means. Since the microfiltration membrane device 110 has a small membrane area, all cells can be viewed in one field of a microscope, and thus, the cells can be analyzed quickly without providing a microscope moving platform.

It should be noted that, for the convenience of describing the core steps of the method of the present invention, some parts of the second apparatus are omitted in fig. 2, but in the design and manufacturing process of the actual device, a reagent flow control unit, a process control system, a man-machine interface, a device housing and corresponding monitoring and protection devices, etc. should be included as required.

It should be noted that the second device shown in FIG. 2 is only an example of a scheme for processing the cells in the microfiltration membrane device 110, and in the specific implementation, the type, amount and sample feeding manner of the reagents are determined and adjusted according to the scheme requirement. The number of the reagents may be 2, 3, 4, 5, 6, 7, 8, 9 or 10.

FIG. 3 is a more specific embodiment of the system of FIG. 2, illustrating one possible implementation of the system when different types of building block functional units are used. The microfluidic cell sorting device 120 shown in fig. 3 is a deterministic laterally offset microfluidic chip; the deterministic lateral-offset microfluidic chip comprises a deterministic lateral-offset region; one side of the deterministic lateral offset region contains an inlet a and the other side contains an outlet B; the inlet A comprises at least two channels, one channel is used for inputting samples, and the other channel is used for inputting buffer solution; the outlet B comprises at least two channels; one channel is used for the outflow of target cells and buffer solution, and the other channel is used for the outflow of sample waste liquid; in one particular application, the ratio of sample waste to filtered recovery flow after exiting the deterministic lateral offset zone may be 1: 1. 2: 1. 3: 1. 4: 1. 5: 1. 6: 1, etc.;

the deterministic lateral offset zone comprises a plurality of guide posts (not shown) arranged in an array and inclined at an angle to the flow direction of the sample; in one embodiment, the spacing between adjacent guide posts (not shown) may be 50 μm + -5 μm; the target cells 250 are slightly shifted in the fixed direction when contacting the guide column (not shown), the target cells 250 are separated from other components in the sample and discharged from the channel B160 with the buffer solution, and the rest of the sample waste 230 is discharged from the channel a 150;

specifically, the sorting system uses a syringe pump to drive to suck the sample 210 to be processed in the sample container 170 to be processed into a syringe of the syringe pump and to deliver the sample to be processed to the microfluidic cell sorting device 120 through the sample injection line 130 according to a set flow rate. The switching between the aspiration of the cell suspension sample into the syringe and the pumping out to the microfluidic cell sorting device 120 is controlled by the second switching valve 440. Meanwhile, the sorting system sucks the buffer solution 220 in the buffer solution container 180 into an injector of the injection pump in a mode of driving the injection pump and conveys the buffer solution to the microfluidic cell sorting device 120 through the buffer solution pipeline 140 according to a set flow rate so as to ensure that a sample and the buffer solution enter the microfluidic cell sorting device 120 according to a certain proportion, thereby realizing the sorting and enrichment of cells. The switching between the buffer solution suction syringe and the buffer solution pump out to the microfluidic cell sorting device 120 is controlled by the third switching valve 450. The recovery solution processed by the microfluidic cell sorting device 120 enters the microfiltration membrane device 110 through the pipe B160. The target cells 250 are fully enriched in the microfiltration membrane device 110 after sample processing is complete.

When the enrichment of the target cells 250 is performed, the sample 210 to be processed containing the target cells 250 flows into the microfluidic cell sorting device 120 through the sample injection line 130, the target cells 250 are separated from other components of the sample in the microfluidic cell sorting device 120 under the action of the buffer solution 220 introduced through the buffer solution line 140, the sample waste solution 230 flows into the waste solution container 190 through the pipeline a150, the target cells 250 flow out of the sample pretreatment system 100 through the enrichment outflow pipeline B160 and enter the microporous membrane device 110, because the membrane pore size of the microporous membrane device 110 is smaller than the diameter of the target cells 250, the target cells 250 are trapped in the microporous membrane device 110, and other recovery solutions and some solid particle components in the sample smaller than the target cells 250 flowing into the pipeline B160 along with the target cells 250 flow into the recovery solution container 200 through the pores of the microporous membrane device 110.

When all of the samples 210 to be processed have been sorted by the microfluidic cell sorting device 120, all of the target cells 250 in the samples 210 to be processed will be retained above the filter by the microporous filter membrane device 110. The reagent switching valve 270 of the second device 360 switches the flow path of the channel B160 to communicate with the second device 360.

Specifically, the second apparatus 360 further includes a reagent switching valve 270, a temperature control system 280, a reagent sampling apparatus 300, a reagent switching apparatus 310, an air filtering apparatus 430, and five reagent loading units composed of a reagent 320, a reagent container 330, a reagent sampling pipeline 340, and the like. The five reagent loading units, the air filtering device 430 and the reagent sampling device 300 are all connected with the reagent switching device 310; wherein the filtering precision of the air filtering device 430 is 0.2 micron; the reagent sampling device 300 can be a peristaltic pump, the inner diameter of a pump pipe of the peristaltic pump is 0.5 mm, and the flow control range is 10-100 microliters per minute.

As shown in fig. 3, after the reagent switching valve 270 switches the flow path of the channel B160 to communicate with the second device 360, the reagent sampling device 300 sequentially loads five reagents 320 into the microporous filter membrane device 110 according to a planned sequence by switching of the reagent switching device 310, thereby performing processes such as fixation, permeabilization, sealing, staining, and washing of cells in the microporous filter membrane device 110. When the temperature in the microfiltration membrane device 110 needs to be controlled after loading some reagents, the temperature in the microfiltration membrane device 110 is changed by the temperature control system 280 by controlling the temperature of the pipe B160 and by heat conduction. When the reagent in the microfiltration membrane device 110 needs to be emptied, the reagent switching device 310 connects the reagent sampling device 300 with the air filtration device 430, and the reagent in the microfiltration membrane device 110 is emptied under the action of the peristaltic pump. The sample in the microfiltration membrane device 110 treated by the second device 360 may be taken by separating the microfiltration membrane device 110 from the line B160 and analyzed under a microscope or by other analysis means for analyzing the cells in the microfiltration membrane device 110. Since the microfiltration membrane device 110 has a small membrane area, all cells can be viewed in one field of a microscope, and thus, the cells can be analyzed quickly without providing a microscope moving platform. When the cell treatment is carried out, the step of frequent centrifugal cleaning required by conventional treatment is avoided, the cleaning time is saved, and the loss and damage to the cells can be reduced to the maximum extent.

If the cells are to be subjected to PCR (polymerase chain reaction analysis) or NGS (high throughput sequencing), the residual liquid in the microporous filter membrane device 110 is absorbed by the filter paper from the bottom, and then cell lysis solution is added to lyse the cells. The above procedure also avoids the need for centrifugation volume concentration of the cells.

It should be noted that, for convenience of describing the core steps of the method of the present invention, the cell centralized sorting system may further include a sample and buffer input and flow control unit, a process control system, a human-machine interface, a device housing, and corresponding monitoring and protection devices, etc. as required during the design and manufacturing process of the actual device.

One specific application may be the enrichment and automated immunofluorescent staining of circulating tumor cells in extracorporeal blood. For example, the sample pretreatment system 100 needs to be pre-filled with buffer solution, which includes steps of adding 7.5 ml of human peripheral blood into the sample container 170 to be treated, adding 50ml of 1 × PBS buffer solution into the buffer container 180, switching the third switching valve 450 to a state where the reagent buffer sample pump 420 is communicated with the buffer line 140, and sucking the buffer solution into the syringe of the reagent buffer sample pump 420 at a flow rate of 30 ml per minute; the second switching valve 440 is switched to the state where the sample pump 260 is in communication with the sample injection line 130, and the third switching valve 450 is switched to the state where the reagent buffer sample pump 420 is in communication with the buffer line 140, so that the buffer solution is filled in the microfluidic cell sorting device 120 through the buffer line 140 at a flow rate of 500 microliters per minute, and the buffer solution is maintained for 1 minute. During the process of using the buffer solution to pre-charge the tube system by the reagent buffer solution sample pump 420, the sample pump 260 first sucks a part of the buffer solution at a flow rate of 100 microliters per minute, and then switches the second switching valve 440 to a state where the sample pump 260 is communicated with the sample container 170 to be processed, and discharges a part of the buffer solution at a flow rate of 100 microliters per minute, so that all the tubes and the gas in the syringe in the sample pre-processing system 100 are discharged, thereby completing the pre-charging. Next, the sample needs to be loaded into the syringe of the sample pump 260, and the steps include sucking all the sample solution at a flow rate of 10 ml per minute while ensuring that no gas is sucked into the syringe, stopping the sample pump 260 and the reagent buffer sample pump 420, and switching the sample pipeline to the state where the sample pump 260 is communicated with the sample injection pipeline 130 by switching the second switching valve 440. After the preparation process is finished, the sample can be processed, and the process includes starting the sample pump 260 and the reagent buffer sample pump 420 at the same time, injecting the cell suspension sample and the buffer into the microfluidic cell sorting device 120 at a flow rate of 0.2 ml/min and 0.5 ml/min, separating the circulating tumor cells in the cell suspension sample under the action of deterministic lateral shift, and intercepting the circulating tumor cells in the microporous membrane device 110 under the action of the microporous membrane device 110. When the cell suspension sample in the sample pump 260 is processed, the inflow of the sample pump 260 is first stopped, the reagent buffer sample pump 420 is continuously pumped into the buffer for about 1 minute, and the reagent buffer sample pump 420 is stopped after all the residual cell suspension samples in the microfluidic cell sorting device 120 and the pipeline a150 are discharged, so that the sorting is completed.

Specifically, five reagent containers 330 each contain 1 XPBS buffer, 4% paraformaldehyde as a fixative, 0.4% Triton X-100 as a permeabilizing agent, blocking solution, and DAPI/CD45/CK cocktail reagent for fluorescent staining. When the reagent switching valve 270 switches the flow path of the tube B160 to communicate with the second device 360, the reagent switching device 310 first switches to a state where the tube B160 communicates with the reagent container 330 containing 1 XPBS buffer, the 1 XPBS buffer is loaded into the microfiltration membrane device 110 at a flow rate of 0.1 ml/min by the reagent sampling device 300, the loading is stopped after washing for 2 minutes, then the reagent switching device 310 switches to a state where the tube B160 communicates with the reagent container 330 containing 4% paraformaldehyde of the fixative solution, the fixative solution is loaded into the microfiltration membrane device 110 at a flow rate of 0.1 ml/min by a peristaltic pump, the loading is stopped after 10 seconds of continuous loading, and the loading is maintained for ten minutes to sufficiently fix the cells, prevent autolysis and cell decay of the cells caused by proteolytic enzymes, enhance the hardness and mechanical strength of the cells, and then the reagent switching device 310 switches again to a state where the tube B160 communicates with the reagent container 330 containing 1 XPBS buffer, loading 1 XPBS buffer solution into the microfiltration membrane device 110 by a peristaltic pump at a flow rate of 0.1 ml per minute, continuously washing for 2 minutes, washing off excessive fixing solution, stopping the loading, then switching the reagent switching device 310 to a state that the pipeline B160 is communicated with a reagent container 330 containing 0.4% Triton X-100 of a permeabilizing agent, loading the permeabilizing agent into the microfiltration membrane device 110 by the peristaltic pump at a flow rate of 0.1 ml per minute, stopping the loading after continuously loading for 10 seconds, keeping for ten minutes to perform membrane rupture permeabilization treatment on cells, perforating a pore channel on cell membranes to allow antibodies or dyes to easily enter and contact intracellular proteins, then switching the reagent switching device 310 to a state that the pipeline B160 is communicated with the reagent container 330 containing 1 XPBS buffer solution again, loading the 1 XPBS buffer solution into the microfiltration membrane device 110 by the peristaltic pump at a flow rate of 0.1 ml per minute, continuously washing for 2 minutes, stopping loading after washing off excessive permeabilizing agent, then switching the reagent switching device 310 to a state that the pipeline B160 is communicated with the reagent container 330 filled with sealing liquid, loading the sealing liquid into the microfiltration membrane device 110 at a flow rate of 0.1 milliliter per minute by a peristaltic pump, stopping loading after continuously loading for 10 seconds, and keeping for thirty minutes to reduce non-specific binding of antibody or dye and a sample in the subsequent dyeing process, then switching the reagent switching device 310 to a state that the pipeline B160 is communicated with the reagent container 330 filled with 1 XPBS buffer solution again, loading the 1 XPBS buffer into the microfiltration membrane device 110 at a flow rate of 0.1 milliliter per minute by the peristaltic pump, continuously washing for 2 minutes, stopping loading after washing off excessive sealing liquid, then switching the reagent switching device 310 to a state that the pipeline B160 is communicated with the reagent container 330 filled with cocktail fluorescent staining reagent, the peristaltic pump loads the cocktail fluorescent staining reagent into the microfiltration membrane device 110 at a flow rate of 0.1 ml per minute, the loading is stopped after continuously loading for 10 seconds, and the loading is kept for sixty minutes to perform the fluorescent staining treatment on the cells in the microfiltration membrane device 110, then the reagent switching device 310 switches the state of the pipeline B160 to be communicated with the reagent container 330 filled with the 1 XPBS buffer solution, the peristaltic pump loads the 1 XPBS buffer into the microfiltration membrane device 110 at a flow rate of 0.1 ml per minute, the washing is continuously performed for 2 minutes, the loading is stopped after the excess cocktail fluorescent staining reagent is washed away, and the fixing, permeabilization, sealing and staining treatment on the cells in the microfiltration membrane device 110 are completed.

The processed sample of circulating tumor cells can be analyzed and identified by separating the microfiltration device 110 from the sample pretreatment system 100 and placing it under an inverted fluorescence microscope.

The operation in the application example avoids frequent centrifugal cleaning steps, realizes automation of the dyeing analysis processing flow, obviously shortens the time required by the process, and simultaneously reduces cell loss caused by the centrifugal cleaning process. On the other hand, because the filtering membrane area of the microporous filtering membrane device 110 is small, cells on the membrane can be conveniently browsed, observed and searched for circulating tumor cells under an inverted fluorescence microscope. The presence of the microfiltration device 110 also allows for the filtration of small amounts of blood cells from the microfluidic pores in the sample pretreatment system 100 into the channel B160 in the event of unexpected flow fluctuations, etc., in the sample pump 260 and the reagent buffer sample pump 420.

Example two

This example provides a cell concentration sorting system, as shown in FIG. 4. FIG. 4 is a more specific embodiment of the system shown in FIG. 2, illustrating another possible way of presenting the system when different types of component functional units are used. Comprises a pretreatment system 100 and a second device 360, wherein the microfluidic cell sorting device 120 further can be a spiral flow channel inertial focusing microfluidic chip; the spiral flow channel inertial focusing micro-fluidic chip comprises a plurality of circles of flow channels which are spirally arranged; one end of the flow channel comprises an inlet C for inputting the diluted cell suspension sample, and the other end of the flow channel comprises an outlet D; the outlet D comprises at least two channels, one channel is used for the target cells and the buffer solution to flow out, and the other channel is used for the sample waste solution to flow out; in a specific application, the flow ratio of the sample waste liquid and the filtered recovery liquid after flowing out of the spiral flow channel can be 1: 1. 2: 1. 3: 1. 4: 1. 5: 1. 6: 1, etc.;

the curvature radius of the spiral flow channel is 5-9 mm, the cross section of the spiral flow channel is trapezoidal, the inner side of the spiral flow channel is 80 micrometers deep, the outer side of the spiral flow channel is 130 micrometers deep, and the width of the spiral flow channel is 600 micrometers wide.

The microfiltration membrane of the microfiltration membrane device 110 is circular with a diameter of 1.5 mm, a thickness of 15 μm, a micropore diameter of 7 μm, and is regularly arranged, with a porosity of 80%.

And the spiral flow channel inertial focusing microfluidic chip is used for shunting the cell suspension sample. The sorting system adopts a constant-pressure driving mode to convey the cell suspension sample in the sample pretreatment system 100 to the spiral flow channel inertial focusing microfluidic chip through the sample introduction pipeline 130. Wherein the start and stop of the delivery can be controlled by the sample injection valve 370. The pressure in the sample container 170 to be processed is regulated by the sample pump 260 and the pressure stabilizer 380, and the pressure stabilizer 380 is a pressure controller; the flow in the sample feeding pipeline 130 and the spiral flow channel inertial focusing micro-fluidic chip meets the sorting requirement.

In fig. 4, a filter 390 for removing solid particles in the gas in the sample container 170 to be processed is further connected to one side of the sample container 170 to be processed. The filtration precision of the filter 390 was 0.2. mu.m.

The recovery solution processed by the microfluidic cell sorting device 120 enters the microfiltration membrane device 110 through the pipe B160. After the target cells are completely concentrated in the microporous filter membrane device 110 after the sample processing is completed, the reagent switching valve 270 of the second device 360 switches the flow path of the channel B160 to communicate with the second device 360.

The microfluidic cell sorting device 120 may also be a micro-vortex microfluidic cell sorting device or a cross-flow filtration (also referred to as tangential flow filtration) chip or other prior art.

Specifically, as shown in fig. 4, the second apparatus 360 includes a reagent switching valve 270, a temperature control system 280, and three reagent loading units including a reagent 320, a reagent container 330, a reagent injection line 340, a reagent injection pump 400, and a first switching valve 410. The first switching valve is mainly used for switching the sample injection state and the supplement state of a sample injection pump of a reagent for processing cells; wherein the injection capacity of the reagent injection pump 400 is 1mL, the flow control range is 10-1000 μ L/min, and all the pipelines are PEEK pipes with the inner diameter of 0.5 mm.

After the reagent switching valve 270 switches the flow path of the channel B160 to communicate with the second device 360, the three reagent loading units are respectively switched to a state where the reagent injection pipeline 340 is communicated with the reagent injection pump 400 through the first switching valve 410, the reagent 320 is replenished into the injector of the reagent injection pump 400 from the reagent container 330 through the reagent injection pipeline 340, and then the first switching valve 410 is switched to a state where the channel B160 is communicated with the reagent injection pump 400, so that the reagent is loaded into the microporous membrane device 110 by the reagent injection pump 400, thereby realizing the treatments such as fixation, permeabilization, sealing, staining, washing, and the like of the cells in the microporous membrane device 110. When the temperature in the microfiltration membrane device 110 needs to be controlled after loading some reagents, the temperature in the microfiltration membrane device 110 is changed by the temperature control system 280 by controlling the temperature of the pipe B160 and by heat conduction. The sample in the microfiltration membrane device 110 treated by the second device 360 may be taken by separating the microfiltration membrane device 110 from the line B160 and analyzed under a microscope or by other analysis means for analyzing the cells in the microfiltration membrane device 110. Since the area of the filter membrane of the microfiltration membrane device 110 is small, all cells can be browsed under one microscope, and thus the cells can be analyzed quickly without configuring a microscope moving platform.

As shown in FIG. 4, another specific application of the second device can be to enrich circulating tumor cells in human peripheral blood from which erythrocytes have been removed and to perform an automated pre-immunofluorescent staining pretreatment. For example, 5ml of 1 × PBS buffer solution is added into the sample container 170 to be processed, the container is sealed, the sample injection valve 370 is closed, the sample injection pump 260 is opened, and the pressure in the sample container 170 to be processed is adjusted to 0.1 mpa under the action of the pressure stabilizer 380. And opening the sample injection valve 370 for 2 minutes, filling the microfluidic cell sorting device 120 and the pipeline A150 and the pipeline B160 with buffer solution, and then closing the sample injection valve 370. After 7.5 ml of human peripheral blood was subjected to erythrocyte lysis, it was resuspended to 15 ml with 1 × PBS buffer, transferred into the sample container 170 to be treated, and the sample container 170 to be treated was closed. The sample pump 260 is turned on again, and the pressure in the sample container 170 to be processed is adjusted to 0.085 mpa under the action of the pressure stabilizer 380, so that the cell suspension sample completely passes through the microfluidic cell sorting device 120 at a flow rate of 1.7 ml per minute. Under the action of inertial focusing and dean vortex, when the cell suspension sample flows to the end of the spiral flow channel, the microfluidic cell sorting device 120 will gather the circulating tumor cells inside the shallow flow channel, and enter the pipeline B160 through the recovery flow channel, the microporous membrane device 110 will intercept the cells therein, and the liquid components will be discharged into the recovery liquid container 200; meanwhile, the white blood cells and the residual red blood cells in the sample are gathered at the outer side of the deeper flow channel, enter the pipeline A150 through the waste liquid flow channel, and are finally collected by the waste liquid container 190.

Specifically, three reagent containers 330 each hold 1 × PBS buffer, a fixative 4% paraformaldehyde, and a permeabilization-blocking premix consisting of 5% goat serum, 5% FcR blocking reagent, 0.4% Triton X-100, and 0.3M glycine in 1 × PBS. When the reagent switching valve 270 switches the flow path of the channel B160 to communicate with the second device 360, first the three reagent loading units are respectively switched to the connection state of the reagent injection pipeline 340 and the reagent injection pump 400 through the first switching valve 410, the reagent 320 is replenished into the injector of the reagent injection pump 400 from the reagent container 330 through the reagent injection pipeline 340, then the first switching valve 410 is switched to the connection state of the channel B160 and the reagent injection pump filled with 1 × PBS buffer, the 1 × PBS buffer is loaded into the microfiltration membrane device 110 at a flow rate of 0.1 ml per minute by the reagent injection pump, the loading is stopped after 2 minutes of washing, then the first switching valve 410 is switched to the connection state of the channel B160 and the injection pump filled with 4% paraformaldehyde of the stationary liquid, the stationary liquid is loaded into the microfiltration membrane device 110 at a flow rate of 0.1 ml per minute by the reagent injection pump, the loading is stopped after 10 seconds of continuous loading, and maintained for ten minutes to sufficiently fix the cells, after which the 1 XPBS buffer is loaded into the microfiltration membrane device 110 again at a flow rate of 0.1 ml per minute by the reagent injection pump, after washing is continued for 2 minutes, the loading is stopped, then the first switching valve 410 is switched to a state that the pipeline B160 is communicated with a reagent injection pump filled with the permeabilization-blocking premix, the permeabilization-blocking premix is loaded into the microfiltration membrane device 110 by the reagent injection pump at a flow rate of 0.1 ml per minute, the loading is stopped after continuous loading for 10 seconds and is kept for thirty minutes, to rupture and block the cells, and then the 1 XPBS buffer solution is loaded into the microfiltration device 110 again at a flow rate of 0.1 ml per minute by the reagent syringe pump, after washing is continued for 2 minutes, the loading is stopped, and the fixation and permeabilization blocking treatment of the cells in the microfiltration membrane device 110 is completed.

The treated sample of circulating tumor cells can be stained by separating the microfiltration device 110 from the sample pretreatment system 100, further soaking in a 96-well plate containing cocktail of fluorescent stains for 1 hour, and removing excess stain by multiple rinses in 1 x PBS buffer to complete the staining of the sample. The stained cell sample can be analyzed and identified under an inverted fluorescence microscope. Because the time for performing fluorescent staining is long, separating the microfiltration device 110 for independent staining can save a lot of processing time by parallel processing of multiple samples while minimizing manual steps.

The operation in the application example avoids frequent centrifugal cleaning steps, realizes automation of the dyeing analysis processing flow, obviously shortens the time required by the process, and simultaneously reduces cell loss caused by the centrifugal cleaning process. On the other hand, because the filtering membrane area of the microporous filtering membrane device 110 is small, cells on the membrane can be conveniently browsed, observed and searched for circulating tumor cells under an inverted fluorescence microscope.

In this embodiment, the time taken for the sample pretreatment system 100 to sort and enrich the sample is within ten minutes, and although the second device 360 performs the fixation and permeabilization sealing treatment on the enriched target cells, the time is saved compared with the scheme of using a centrifuge to clean, and the flow of processing one sample still exceeds 45 minutes. When more samples need to be processed, the sample preprocessing system 100 can be operated separately and independently from the second device 360.

As shown in fig. 5B, the second apparatus 360 may further include a plurality of reagent outlet lines 480, reagent dispensing control valves 470 for controlling the outflow of the reagents, reagent dispensing lines 460, a reagent waste liquid collection container 490, and the like; the reagent waste liquid recovery container 490 contains the waste liquid 500 flowing out of the reagent outlet pipe 480. Each reagent outlet line 480 of the second device 360 may be coupled to a microporous membrane device 110 with a cellular sample separated from the sample pretreatment system 100 for performing a staining-related process on the cells therein.

The specific method mainly comprises the steps that the sample pretreatment system 100 without the second device 360 shown in FIG. 5A is used for sorting and enriching the sample, and after the enrichment is completed, the microfiltration membrane device 110 is separated from the sample pretreatment system 100 and is sleeved on the independent second device 360 shown in FIG. 5B. When the second device 360 needs to process more samples at the same time, a plurality of microfiltration membrane devices 110 can be sleeved on the independent second device 360 at the same time, and the reagent distribution control valve 470 is used for controlling the reagent loading of the cells in the microfiltration membrane device 110. The loading process may be performed simultaneously after all the microfiltration membrane devices 110 are connected to the second device 360, that is, the reagent distribution control valves 470 are opened simultaneously to load the same reagents on the cells in the microfiltration membrane devices 110, or may be performed in a time-sharing manner, that is, after the first sample sorting process performed by the sample pretreatment system 100 is completed, the microfiltration membrane devices 110 are separated and connected to the first reagent outlet pipe 480 of the second device 360 in a sleeved manner, and the pre-staining process is started, and at this time, the reagent distribution control valves 470 connected to the other reagent outlet pipes 480 are all in a closed state. After the second sample sorting process performed by the sample pretreatment system 100 is completed, the microfiltration membrane device 110 is separated and connected to the second reagent outlet line 480 of the second device 360, and when the first sample is in a holding state after being loaded with a certain reagent, the reagent dispensing control valve 470 connected to the second reagent outlet line 480 is opened to perform operations such as washing and reagent loading, and so on to the case of three or more samples. Since the hold state is significantly longer than the time required for buffer washing and reagent loading, time-shared sequential processing of multiple samples is easily achieved.

EXAMPLE III

The invention also comprises a cell sorting method, which applies the cell centralized sorting system; as shown in fig. 6, wherein, the following steps are included:

It will be understood that modifications and variations can be made by persons skilled in the art in light of the above teachings and all such modifications and variations are intended to be included within the scope of the invention as defined in the appended claims.

Claims

1. The centralized cell sorting system is characterized by comprising a microfluidic cell sorting device for performing target cell enrichment on a cell suspension sample containing target cells, and a first driving mechanism for driving the cell suspension sample into the microfluidic cell sorting device; the microfluidic cell sorting device is provided with a pipeline A for the sample waste liquid to flow out and a pipeline B for the target cells to flow out; the pipeline B is also provided with a microporous filter membrane device which can be separated from the pipeline B and used for filtering the target cells.

2. The centralized cell sorting system of claim 1, wherein the microfluidic cell sorting device is a deterministic laterally offset microfluidic chip; the deterministic lateral-offset microfluidic chip comprises a deterministic lateral-offset region; one side of the deterministic lateral offset region contains an inlet a and the other side contains an outlet B; the inlet A comprises at least two channels, one channel is used for inputting the cell suspension sample, and the other channel is used for inputting a buffer solution; the outlet B comprises at least two channels; one channel is used for the target cells and the buffer solution to flow out, and the other channel is used for the sample waste solution to flow out.

3. The centralized cell sorting system according to claim 1, wherein the microfluidic cell sorting device is a spiral flow channel inertial focusing microfluidic chip; the spiral flow channel inertial focusing micro-fluidic chip comprises a plurality of circles of flow channels which are spirally arranged; one end of the flow channel comprises an inlet C for inputting the diluted cell suspension sample, and the other end of the flow channel comprises an outlet D; the outlet D comprises at least two channels, one channel is used for the target cells to flow out, and the other channel is used for the sample waste liquid to flow out.

4. The centralized cell sorting system of claim 1, further comprising a second means for automated labeling of the target cells; the second device is communicated with the microporous filter membrane device.

5. The centralized cell sorting system of claim 4, wherein the second device comprises a temperature control system, a reagent switching device, at least one reagent feeding line connected to the reagent switching device, and a second driving mechanism for driving the fluid in the reagent feeding line into the microfiltration membrane device.

6. The centralized cell sorting system of claim 5, wherein the second device further comprises a reagent switching valve for switching the flow of the fluid in the second device into the microfiltration membrane device.

7. The centralized cell sorting system according to claim 1, wherein the microfiltration membrane device comprises a microfiltration membrane and an annular member that fixes the microfiltration membrane to the tube B; the annular member is tightly fitted over the output end of the pipe B.

8. The cell mass sorting system according to claim 7, wherein the pore size of the microporous filter membrane is smaller than the diameter of the target cell.

9. The cell centralized sorting system of claim 8, wherein the porosity of the microporous filter membrane is not less than 20%.

10. A cell sorting method using the centralized cell sorting system according to any one of claims 1 to 9; the method is characterized by comprising the following steps:

step two: introducing the cell suspension sample after being sorted in the step one into a microporous filter membrane device along a fixed flow channel, wherein the microporous filter membrane device filters partial non-target particle components and waste liquid, and simultaneously entraps the target cells in the microporous filter membrane device to finish sorting;