CN107084916B - Circulating tumor cell separation micro-fluidic chip device and application method thereof - Google Patents
Circulating tumor cell separation micro-fluidic chip device and application method thereof Download PDFInfo
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Classifications
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Abstract
The invention relates to a circulating tumor cell separation microfluidic chip device and a using method thereof, the device comprises a microfluidic chip (1) and a chip clamp (2), the microfluidic chip (1) comprises a chip inlet (11), a single-spiral chip (12), rare cell collecting pipelines (13, 14) and a white blood cell outlet (15), a single-spiral channel enters a spiral microfluidic channel from the chip inlet (11) at the center of the circular spiral channel through a semicircular initial channel, and the tail end comprises 3 outlets: respectively collecting rare cells with different particle sizes; the chip clamp (2) is automatically abutted with the inlet and outlet of the micro-fluidic chip (1). Compared with the prior art, the microfluidic chip device can automatically separate and enrich tumor cells in liquid samples such as peripheral blood, pleural effusion, cerebrospinal fluid and the like of tumor patients by using the processed samples, and remarkably improves the accuracy and specificity of the tumor patients in accurate and personalized treatment.
Description
Technical Field
The invention relates to the field of cell sorting, in particular to a device and a method for automatically sorting and circulating tumor cells from a biological liquid sample on a microfluidic chip.
Background
In biomedical fields and the like, it is often necessary to sort out specific target cells from a more complex sample. Such as detecting, sorting and counting the number of circulating tumor cells (CTCs, circulating tumor cell) in the blood of cancer patients, are becoming increasingly important in cancer diagnosis and treatment. The current CTC detection technology, namely fish-dragon hybridization, generally comprises two major types of markers, namely a positive enrichment method and a negative enrichment method based on antibody-coated magnetic beads based on marker capture. Such belongs to the first generation of CTC capture technology, representing the strong Cell search system. However, the technical disadvantage is that antibody capture is limited to capture of epithelial-derived CTCs that specifically express EpCAM epithelial antigen, and that viable cell capture cannot be achieved for subsequent gene detection. The negative enrichment method is used for reversely removing the leucocytes in peripheral blood through CD45 magnetic beads, and cannot carry out enrichment detection on trace CTC in a large-volume sample, and the problem of nonspecific adsorption of the magnetic beads exists. The first-generation technology filter membrane method can only capture tumor cells with large particle size, leak detection of tumor cells with small particle size, and the captured cells have no cell activity and can only be used for cell counting, morphology and FISH detection.
Microfluidic chips are self-evident as a supporting technology for life sciences in the 21 st century, and are a technical core of portable biochemical analysis instruments. The technology integrates basic operation units such as sample preparation, biological and chemical reaction separation and detection related to the fields of biology, chemistry and the like on a chip of a few square centimeters through constructing a micro-scale channel, so as to finish different biological or chemical reaction processes, analyze a large number of biomolecules in a short time, accurately acquire a large amount of information in the sample, and has information quantity hundreds to thousands times that of the traditional detection means.
There have been some studies reported on sorting circulating tumor cells in blood samples on microfluidic chips, such as using dielectrophoresis, hydrodynamic force, immunomagnetic bead-based and fluorescent sorting methods, etc. However, the method has the defects of different degrees, such as the method of direct current dielectrophoresis, which requires the application of higher electric field intensity and is extremely easy to cause cell lysis; although the separation efficiency of tumor cells expressed by specific antigens is improved based on the immunomagnetic bead separation method, the separation method is still limited to the recognition of tumor cell antigen epitopes, and the heterogeneity of circulating tumor cells determines the capture method based on antigen recognition by adopting a single antibody or a plurality of antibodies, so that the problem of missed detection of non-antigen expressed CTCs exists. The fluorescence sorting method needs to perform fluorescence labeling on cells, and needs to build a complex optical detection system.
Patent 201410336948.4 discloses a PDMS microfluidic chip for separating circulating tumor cells, which comprises a liquid storage hole A to a liquid storage hole F, a detection channel, a main channel, a first focusing channel, a second focusing channel, a sample outlet channel, a target cell collecting channel and an electromagnetic sorting channel; when the circulating tumor cells pass through the detection channel, the generated voltage signals are detected and sent to the NI acquisition card and the processing terminal, the processing terminal sends out the voltage signals through the NI acquisition card, so that the electromagnetic relay is closed, and then the electromagnetic micro-valve structure presses the PDMS layer below the electromagnetic micro-valve structure to deform, so that a part of liquid is discharged from the liquid storage hole E, and the circulating tumor cells are pushed to flow into the target cell collection channel. The chip device requires an electric field and a magnetic field device to achieve separation of cells. The chip is complex in whole preparation, multiple in operation flow and high in cost.
Patent 201410345305.6 discloses a double-helix microfluidic chip, which comprises a red blood cell outlet, a white blood cell outlet, a fluid outlet and a cell filter membrane, and basically, the filtration and separation of tumor cells are realized by relying on an 8-micrometer aperture filter membrane, and the method still can leak detection of tumor cells with small particle sizes.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a circulating tumor cell separation microfluidic chip device and a use method thereof. Based on the difference of the nuclear-plasma ratio and the surface charge of the circulating tumor cells and the blood cells, the invention adopts the fluid mechanics on a microfluidic chip to realize the separation and enrichment of the circulating tumor cells in the sample which are not dependent on the antibody.
The aim of the invention can be achieved by the following technical scheme: the device comprises a microfluidic chip and a chip clamp, wherein the microfluidic chip comprises a chip inlet, a single-screw chip, a rare cell collecting pipeline and a white blood cell outlet, wherein the rare cell collecting pipeline comprises two rare cell collecting pipelines which are respectively connected with an inner side outlet (13) and an outlet (14); the single-screw chip is composed of a single-screw channel, the single-screw channel enters a spiral microfluidic channel from a chip inlet at the center of a circular screw channel through a semicircular initial channel, and the tail end of the single-screw channel comprises 3 outlets: an outlet (13) and an outlet (14) for collecting rare cells with different particle sizes and a waste liquid outlet (15) for collecting white blood cells;
the chip clamp is automatically abutted to the chip inlet, the rare cell collecting pipeline and the waste liquid outlet of the micro-fluidic chip.
The width of the inner outlet is 110+/-50 mu m, the width of the outlet is 110+/-50 mu m, and the width of the white blood cell outlet is 700+/-100 mu m.
The outlets (13) and (14) of the CTC collecting pipeline are both treated by adopting positive charge polymers for surface coating.
The positive charge polymer is a cationic polymer and comprises an amphiphilic polymer modified by polyethyleneimine and acrylamide.
The single spiral channel is a main channel, the width of the single spiral channel is 500+/-100 mu m, the height of the single spiral channel is 120+/-50 mu m, the distance between each single spiral is 500+/-100 mu m, and the number of complete spirals is more than or equal to 3.
The chip clamp comprises a sample pipe joint, a CTC collecting pipe joint and a waste liquid collecting pipe joint; closing the chip clamp, abutting the sample pipe joint with the chip inlet, abutting the CTC collecting pipe joint with different particle sizes with the outlets (13) and (14), abutting the leukocyte collecting pipe joint with the outlet (15) to enable each inlet and outlet of the microfluidic chip to be connected with an external connection conduit, wherein the other end of the sample pipe joint is used for connecting a biological sample, and the biological sample is injected into the chip device at a constant speed through an injection pump or a pressure pump device, and the flow rate of the biological sample is 50-300ml/h.
The flow rate of the biological sample injected into the chip device at a constant speed is preferably 60-200ml/h.
The flow rate is more preferably 100ml/h,120ml/h or 150ml/h.
The biological sample is peripheral blood, pleural effusion, peritoneal effusion, cerebrospinal fluid, bone marrow fluid and/or urine.
The application method of the circulating tumor cell separation micro-fluidic chip device comprises the following steps:
(1) After a blood sample to be tested is subjected to lysis treatment by using a red blood cell lysate (the red blood cell lysate is a commercially available product, such as a lysate produced and sold by the company of biological medicine, non-tin-nano-o), PBS buffer solution is added to dilute the blood sample to obtain a sample introduction sample;
(2) Injecting the sample injection sample in the step (1) into a chip inlet of the microfluidic chip from a sample pipe joint of the chip clamp through a guide pipe by matching with an injection pump or a pressure pump device, and realizing enrichment and separation of circulating tumor cells in a single-screw chip according to the difference of the nuclear-mass ratio and the surface charge of the cells;
(3) The enriched and separated CTC cells with different particle diameters flow out from outlets (13) and (14), are led out through a CTC collecting pipe joint and an accessory catheter and are connected into a sterile centrifuge tube, and the white blood cells flow out from an outside white blood cell outlet and are led out through a white blood cell collecting pipe joint and an accessory catheter.
The volume ratio of the PBS buffer solution to the blood sample to be measured in the step (1) is 10-30:1.
Compared with the prior art, when the biological sample passes through the microfluidic chip, tumor cells and other cells are focused at different positions from the origin of the center of an inlet by utilizing the inertia effect and Dean flow effect of fluid in a spiral flow channel, tumor cells and white blood cells with different nuclear-matter ratios and surface charges are converged into parallel lines with different positions under the hydrodynamic effect, and in the flow velocity range defined by the invention, the cells can bear different fluid forces due to the difference of the nuclear-matter ratios and the charges, and different cells form different spatial arrangements in a pipeline and finally flow out from different outlets. Wherein, most white blood cells enter the blood cell flow channel after converging into a line, and most tumor cells enter the CTC flow channel respectively through the charge adsorption function in the structural flow channel. The microfluidic chip integrated by the spiral flow passage inertial separation technology structure and the charge adsorption technology overcomes the defects of low integration level, low capture rate and the like of the existing circulating tumor cell separation method, and realizes the rapid high-flux separation of the circulating tumor cells which are not dependent on the tumor cells by antibodies; in addition, the chip of the system has the advantages of simple structure, convenient processing, simple operation, high degree of automation and the like, and can be used in the fields of rare cell biology research, early disease diagnosis and treatment and the like.
Drawings
FIG. 1 is a block diagram of a microfluidic chip of the present invention;
FIG. 2 is a diagram of a chip holder opening configuration of the present invention;
FIG. 3 is a diagram of a chip gripper closure structure of the present invention;
FIG. 4 is a schematic diagram of a microfluidic chip according to the present invention for simulating cell separation;
FIG. 5 is a graph showing the enrichment effect of the microfluidic device of the present invention on GFP-transfected MGC803 cells added to normal blood (300 MGC803 cells were added to each sample, and the recovered MGC803 cells were counted after sorting);
FIG. 6 is an immunofluorescence staining chart of circulating tumor cells in peripheral blood isolated in example 1;
FIG. 7 is a staining pattern of circulating tumor cells Giemsa isolated from the pleural effusion isolated in example 2.
Detailed Description
The invention will now be described in detail with reference to the drawings and specific examples.
Example 1
A circulating tumor cell separation microfluidic chip device comprises a microfluidic chip 1 and a chip clamp 2.
As shown in fig. 1, the microfluidic chip 1 includes a chip inlet 11, a single-screw chip 12, a rare cell collecting channel and a white blood cell outlet 15, wherein the rare cell collecting channel includes two rare cell collecting channels, which are respectively connected with an inner outlet 13 and an outlet 14; the single-screw chip 12 is composed of a single-screw channel, the single-screw channel enters the spiral microfluidic channel from a chip inlet 11 in the center of the circular spiral channel through a semicircular initial channel, and the tail end of the single-screw channel comprises 3 outlets: an inner outlet 13 and an outlet 14 for collecting rare cells of different particle sizes, respectively, and an outer white cell outlet 15 for collecting white cells; wherein the width of the inner outlet 13 is 110+ -50 μm, the width of the outlet 14 is 110+ -50 μm, and the width of the leukocyte outlet 15 is 700+ -100 μm. The single spiral channel is a main channel, the width of the single spiral channel is 500+/-100 micrometers, the height of the single spiral channel is 120+/-50 micrometers, the distance between each single spiral is 500+/-100 micrometers, and the number of complete spirals is more than or equal to 3, in the embodiment, 5 complete spirals.
The inner side outlet 13 and the outer side outlet 14 are both treated with surface coating by positively charged polymer polyethyleneimine. The treatment method is a conventional liquid coating method, namely a natural coating method by adopting a certain volume of polymer solution to be injected into an outlet through a syringe.
As shown in fig. 2 to 3, the chip holder 2 includes a sample tube joint 21, CTC collection tube joints 22, 23, and a white cell collection tube joint 24; closing the chip clamp 2, abutting the sample pipe joint 21 with the chip inlet 11, abutting the CTC collecting pipe joints 22 and 23 with the outlets 13 and 14 respectively, abutting the white blood cell collecting pipe joint 24 with the white blood cell outlet 15 to enable the inlets and outlets of the microfluidic chip 1 to be automatically abutted with the external connecting pipe, wherein the other end of the sample pipe joint 21 is connected with a biological sample, the biological sample is injected into the chip device at a constant speed through an injection pump or a pressure pump device, and the flow rate of the biological sample can be 50-300ml/h.
The biological sample is peripheral blood, pleural effusion, peritoneal effusion, cerebrospinal fluid, bone marrow fluid and/or urine.
The peripheral blood is selected as a biological sample, and the circulating tumor cells in the blood are separated by adopting the circulating tumor cell separation microfluidic chip device, and the method comprises the following steps:
(1) After a blood sample to be tested is subjected to lysis treatment by using a red blood cell lysis solution (Wutanaoao biological medicine Co., ltd.), 15ml of PBS buffer solution is added to dilute the blood sample to be sampled;
(2) After the microfluidic chip 1 and the chip clamp 2 are assembled, the microfluidic chip 1, an external sample liquid tube, an external tube for enriching products, a middle tube and an internal tube are assembled together with a matched catheter, a syringe pump or a pressure pump device as shown in fig. 4;
(3) Starting an instrument, injecting a sample in the step (1) into a chip inlet 11 of the microfluidic chip 1 from a sample pipe joint 21 of the chip clamp 2 through a conduit according to the flow speed of 120ml/h, and realizing enrichment and separation of circulating tumor cells in a single-screw chip 12 according to the difference of the nuclear-plasma ratio and the surface charge; the enriched and isolated CTC cells were discharged from the outlets 13 and 14, led out through the CTC collection tube connectors 22 and 23 and the additional tube, and put into a 5ml sterile centrifuge tube (inner tube), and the white blood cells were discharged from the outside white blood cell outlet 15, led out through the white blood cell collection tube connector 24 and the additional tube, and collected in the outer tube (as shown in fig. 4).
(4) And (3) carrying out throwing on the circulating tumor cells subjected to microfluidic separation, and manufacturing a sample glass slide. 4% paraformaldehyde fixation, permeabilization of cells with 0.25% triton X-100 (PBS formulation) for 10 min, PBS rinse 3 times, 5 min/times; blocking with 2% BSA for 30min, rinsing with PBS for 3 times, and 5 min/time; epCAM (1:200, PBS formulation) was added and incubated in the dark for 2 hours, and PBS was rinsed 3 times, 5 min/time; CK-18 (1:400, PBS) was added and incubated in the dark for 2 hours; rinsing with PBS for three times for 5min each time; adding CD45 (1:200, PBS), incubating in a dark room for 30min, and rinsing with PBS for 3 times and 5 min/time; adding 0.5ug/ml DAPI (PBS for preparation) for dyeing for 10 min, rinsing 3 times by PBS for 5 min/time; adding 20ul of sealing tablet sealing tablets; observed by fluorescence microscopy.
The tumor cells sorted by the microfluidic device of the invention can be observed to have complete cell morphology under a microscope, and the immunofluorescent staining of FIG. 6 shows that the number of EPCAM positive cells is 5, and the nuclear-cytoplasmic ratio is more than 0.8.
As shown in fig. 5, the results of capturing efficiency of the different sample injection rates for separating tumor cells by the microfluidic chip (GFP-positive MGC803 cells were added in an initial amount of 300) indicate that: the average enrichment rate of the nano microfluidic chip at the sample injection speed of 1.0ml/min, 1.5ml/min and 2.0ml/min is 57.7%, 66.3% and 75.2% respectively.
Example 2
The microfluidic device was used to isolate circulating tumor cells in pleural effusion, in the same manner as in example 1, as follows:
(1) The pleural effusion sample is gently turned upside down back and forth to be uniformly mixed; the pleural effusion was diluted 100-fold with PBS and aspirated with a 30ml syringe; then the whole syringe is fixed on a constant flow pump matched with the instrument, the outlet of the syringe is connected with the interface of the microfluidic chip device, and the flow rate of the hydrothorax pump is 130ml/h.
(2) The enriched separation liquid flows out of the enriched separation outlet and is connected into a 5ml sterile centrifuge tube.
(3) And (3) carrying out throwing on the circulating tumor cells subjected to microfluidic separation, and manufacturing a sample glass slide. 4% paraformaldehyde is fixed, giemsa staining is carried out, and a photograph is observed under a microscope, as shown in FIG. 7, and the separated tumor cells show typical cell morphology structures with large nuclei, high nuclear-mass ratio and large particle size.
Example 3
The inner side outlet 13 and the outer side outlet 14 are both treated with a surface coating using a positively charged polymer acrylamide. The biological sample is injected into the chip device at a constant speed by a syringe pump or a pressure pump device, and the flow rate is 50ml/h. The volume ratio of the PBS buffer solution to the blood sample to be measured is 10:1. The procedure is as in example 1.
Example 4
The inner side outlet 13 and the outer side outlet 14 are both treated with a surface coating using a positively charged polymer acrylamide. The biological sample was injected into the chip device at a constant speed by a syringe pump or a pressure pump device at a flow rate of 300ml/h. The volume ratio of the PBS buffer solution to the blood sample to be measured is 30:1. The procedure is as in example 1.
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solution of the present invention should fall within the scope of protection defined by the claims of the present invention without departing from the design spirit of the present invention.
Claims (10)
1. The device is characterized by comprising a microfluidic chip (1) and a chip clamp (2), wherein the microfluidic chip (1) comprises a chip inlet (11), a single-screw chip (12), a rare cell collecting pipeline and a white blood cell outlet (15), and the rare cell collecting pipeline comprises two rare cell collecting pipelines which are respectively connected with an inner side outlet (13) and an outlet (14); the single-screw chip (12) is composed of a single-screw channel, the single-screw channel enters a spiral microfluidic channel from a chip inlet (11) at the center of a circular screw channel through a semicircular initial channel, and the tail end of the single-screw channel comprises 3 outlets: an inner outlet (13) and an outlet (14) for collecting rare cells with different particle sizes and an outer white cell outlet (15) for collecting white cells; the chip clamp (2) is automatically abutted to a chip inlet (11), rare cell collecting pipelines (13, 14) and a white blood cell outlet (15) of the micro-fluidic chip (1), wherein the number of complete spirals of the single spiral channel is more than or equal to 3;
the chip clamp (2) comprises a sample pipe joint (21), CTC collecting pipe joints (22, 23) and a waste liquid collecting pipe joint (24); the chip clamp (2) is closed, the sample pipe joint (21) is in butt joint with the chip inlet (11), the CTC collecting pipe joint (22) is in butt joint with the inner side outlet (13), the CTC collecting pipe joints (23) with different particle diameters are in butt joint with the outlets (14) with different particle diameters, the waste liquid collecting pipe joint (24) is in butt joint with the outlets (15), so that each inlet and outlet of the microfluidic chip (1) are connected with an external connection pipe, and the other end of the sample pipe joint (21) is used for connecting biological samples.
2. The microfluidic chip device for separating tumor cells according to claim 1, wherein the width of the inner outlet (13) is 110+ -50 μm, the width of the outlet (14) is 110+ -50 μm, and the width of the white blood cell outlet (15) is 700+ -100 μm.
3. The circulating tumor cell separation microfluidic chip device according to claim 1, wherein the inner side outlet (13) and the outlet (14) are both surface-coated with positively charged polymers.
4. The circulating tumor cell separation microfluidic chip device according to claim 3, wherein the positively charged polymer is a cationic polymer comprising an amphiphilic polymer modified with polyethylenimine or acrylamide.
5. The device of claim 1, wherein the single spiral channel is a main channel, the width of the single spiral channel is 500±100 μm, the height of the single spiral channel is 120±50 μm, the pitch of each single spiral is 500±100 μm, and the number of complete spirals is 3 or more.
6. The circulating tumor cell separation microfluidic chip device according to claim 1, wherein the other end of the sample tube joint (21) is used for connecting a biological sample, and the biological sample is injected into the chip device at a constant speed through a syringe pump or a pressure pump device, and the flow rate of the biological sample is 50-300ml/h.
7. The circulating tumor cell separation microfluidic chip device of claim 6, wherein the flow rate of the biological sample injected into the chip device at a constant rate is implemented at 60-200ml/h.
8. The microfluidic chip device for separating tumor cells according to claim 6, wherein the flow rate is 100ml/h,120ml/h or 150ml/h, and the biological sample is peripheral blood, pleural effusion, peritoneal effusion, cerebrospinal fluid, bone marrow fluid and/or urine.
9. A method of using the circulating tumor cell separation microfluidic chip device of any one of claims 1-8, comprising the steps of: (1) After the blood sample to be measured is subjected to lysis treatment by using a red blood cell lysate, PBS buffer solution is added, and the sample is diluted into a sample injection sample; (2) Injecting the sample injection sample in the step (1) into a chip inlet (11) of the microfluidic chip (1) from a sample pipe joint (21) of the chip clamp (2) through a guide pipe by matching with a syringe pump or a pressure pump device, and realizing enrichment and separation of circulating tumor cells in a single-screw chip (12) according to the difference of the nuclear-plasma ratio and the surface charge; (3) The enriched and separated CTC cells flow out from an inner outlet (13), are guided out through a CTC collecting pipe joint (22) and an accessory catheter and are connected into a sterile centrifuge tube, CTC with different particle sizes flow out from an outlet (14), are guided out through a collecting pipe joint (23) and an accessory catheter, and white blood cell samples flow out from an outlet (15) and are guided out through a collecting pipe joint (24) and an accessory catheter.
10. The method of claim 9, wherein the volume ratio of the PBS buffer solution to the blood sample to be measured in the step (1) is 10-30:1.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710195836.5A CN107084916B (en) | 2017-03-29 | 2017-03-29 | Circulating tumor cell separation micro-fluidic chip device and application method thereof |
| CN202311503314.9A CN117511696A (en) | 2017-03-29 | 2017-03-29 | Circulation tumor cell separation micro-fluidic chip device |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN201710195836.5A CN107084916B (en) | 2017-03-29 | 2017-03-29 | Circulating tumor cell separation micro-fluidic chip device and application method thereof |
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| WO2019117800A1 (en) * | 2017-12-11 | 2019-06-20 | A. Menarini Biomarkers Singapore Pte Ltd | Microfluidic device and method for cell separation of a blood sample |
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