CN216639471U - Particulate matter capturing system - Google Patents

Abstract

The invention relates to a capture system of particulate matter, which belongs to the technical field of liquid biopsy, and comprises: the centrifugal tube comprises a centrifugal tube body and a membrane separation assembly, the centrifugal tube body is provided with a hanging piece, the inner cavity of the centrifugal tube is provided with an assembly installation cavity and a centrifugate containing cavity, the centrifugate containing cavity is arranged at one end far away from the hanging piece, and the membrane separation assembly is arranged in the assembly installation cavity; on the basis of the existing fluid auxiliary separation, the design of an assembly device for centrifugal membrane filtration by using a horizontal angle rotor solves the problems that particulate matters (such as cells) are stressed at the inner side and the outer side of a membrane in different sizes during centrifugation, are easy to accumulate at the edge to cause blockage, and influence the membrane filtration efficiency and the cell survival.

Description

Particulate matter capturing system

Technical Field

The invention belongs to the field of liquid biopsy, and particularly relates to a particulate matter capturing system.

Background

Enrichment of Circulating Tumor Cells (CTCs) utilizes differences between tumor cells and other blood constituent cells, including differential expression of cell surface proteins or cell unique physical properties. The method mainly comprises the following steps: biological based immunocapture and physical based large separation (e.g.: membrane filtration);

in the affinity-based immunocapture method, CTCs are positively selected using epithelial surface antigens (cell adhesion molecules EpCAM, epidermal growth factor receptor EGFR and mucin 1), mesenchymal antigens (mesenchymal vimentin) and tissue-specific antigens (including prostate-specific membrane antigen PSMA against prostate cancer cells and HER2 against breast cancer cells), in addition cytokeratin CK and N-cadherin; CTCs negatively gate the antibody to recognize leukocyte surface antigens (usually CD45) and other cell surface antigens circulating in the blood (e.g., endothelial cell antigen CD146, hematopoietic stem cell antigen CD34) to remove non-tumor cells. Label-free detection based on cell physical properties exploits differences in cell physical characteristics (e.g., size, shape, density, deformability, adhesiveness, etc.), liquid separation media can centrifugally enrich CTCs according to cell density, microfiltration techniques allow blood to capture CTCs through pores or microfluidic steps, microfluidic devices use inertial focusing to separate CTCs from other components in blood, and Dielectrophoresis (DEP) can separate CTCs according to the unique charges of tumor cells and blood cells.

Affinity-based approaches can use specific biochemical markers to capture CTCs, showing good specificity, but are largely dependent on the expression levels of cell surface biomarkers, cancer cell heterogeneity and epithelial-mesenchymal transition can lead to loss of CTC detection, while the immunolabeling of CTCs is likely to affect cell viability, and furthermore, antibody-based approaches are often expensive and time consuming. The physical property-based method has advantages of simplicity, rapidity, high throughput, etc., and can obtain intact CTCs without being processed by labeling, etc., wherein a method of capturing CTCs according to cell size and deformability is widely studied. The flow line or filtration method based on microchip can reach high flow rate, but usually has the problems of easy blockage, sensitive flow rate, poor robustness and the like, and the method based on membrane filtration or centrifugation is simple, low in cost, convenient to operate, capable of achieving high flux and high recovery rate, and easy to automate and commercialize.

Membrane filtration typically suffers from clogging, high pressure drop and impaired cell viability. Kang et al propose a tapered slit film filter made of a photopolymer for separating CTCs from a blood sample of a cancer patient. The filter has a wider cell inlet and a narrowing outlet, which reduces the pressure to capture cells, and the microfilter is inserted into the filter cassette and simply connected to a commercial syringe for CTC capture during use. Different types of cancer cells are separated by using a filter, the capture rate is 77.7 +/-10.0%, and the cell viability is 80.6%. The filter membrane is generally manufactured using a complicated manufacturing process such as electron beam lithography or reactive ion etching, which may limit its application.

The traditional single-layer filtering membrane easily generates high mechanical stress on cells and damages cell membranes. Zhou et al designed a separable double layer (SB) microfiltration device with a gap between the upper and lower porous membranes. The parylene-C pore edges at the top of the filter capture large size CTCs, medium size leukemia (WBCs) are captured between the two membranes, and small size Red Blood Cells (RBCs) flow out of the device. In use, the microfilter is held between two sealing O-rings formed from Polydimethylsiloxane (PDMS) and sandwiched within a plastic housing. The top of the housing box is connected to the sample loading syringe, while the bottom of the housing box is integrated with the waste collector with a luer fitting. The capture rate of the CTC of the SB microfiltration device is 78-83%, the cell survival rate is 71-74%, and the enrichment is 1.7-2 × 10 relative to the leukocyte³And (4) doubling.

In order to mass produce membrane filters at very low cost, Bu et al propose a fabric filter produced with polyester monofilament yarns for CTC separation. When in use, the fabric piece is cut into a circle with the diameter of 20mm, and the fabric piece is connected through a clamp to form a filter and then can be connected with a commercial syringe. The cell with the curved wall filter can reduce 21.6% mechanical stress compared to the conventional straight wall cell, retain cell viability when capturing CTCs using the fabric filter, capture over 84% of viable cells.

To achieve high throughput membrane filtration, Liu et al designed a 2.5D microporous array filter membrane with a porosity > 40.2% for capturing rare tumor cells. The filtration membrane was prepared using Parylene C molding technology and the prepared membrane was assembled with a self-designed polytetrafluoroethylene (Teflon) holder and a commercially available Nd-Fe-B strong ring magnet into a filtration device prior to filtration. When an undiluted whole blood sample is analyzed only by gravity driving, the filtration flux can reach 17mL/min, and the capture rate of A549 cells in the whole blood is 83.2 +/-6.2%.

The membrane filtration method using centrifugation enables simple, high-throughput CTC capture. Kim et al propose a Fluid Assisted Separation Technique (FAST) for size-selective CTC separation through membrane pores filled with a stabilizing liquid throughout the filtration process, which can reduce the pressure drop by 50% and achieve a milder and more efficient filtration. The microfluidic layout of the FAST disc consists of three independent filtration units, each unit having three chambers for sample injection, filtration and storage of liquids. The polycarbonate membrane was etched using a track with 8 μm pores during filtration, and the discs were rotated at a lower speed (600rpm) to achieve 95.9% recovery of CTCs and 2.5-log removal of WBCs at a throughput of 3 mL/min.

Disclosure of Invention

The application aims to provide a particulate matter (such as cancer cells) capturing system to solve the problems of low particulate matter separation efficiency, easy blockage, complex operation, high cost and low survival rate of living particulate matter (such as cancer cells) caused by the existing membrane filtration.

The embodiment of the invention provides a cancer cell capturing system, which comprises:

the centrifugal tube comprises a centrifugal tube body and a membrane separation assembly, the centrifugal tube body is provided with a hanging piece, an assembly installation cavity and a centrifugate containing cavity are formed in the inner cavity of the centrifugal tube, the centrifugate containing cavity is formed in one end far away from the hanging piece, and the membrane separation assembly is installed in the assembly installation cavity;

the horizontal rotor comprises a rotating part and a hanging part, the rotating part is rotatably arranged on the working platform, and the hanging part is connected to the rotating part; the hanging part is used for hanging the hanging part so as to realize centrifugal separation of the centrifugal tube.

Optionally, the membrane separation module comprises: subassembly, separation membrane and a sample adding section of thick bamboo are placed to the membrane, the separation membrane is located in the subassembly is placed to the membrane, add a sample section of thick bamboo connect in subassembly one end is placed to the membrane.

Optionally, the membrane separation module further comprises: the liquid hold up tank, the recess end of liquid hold up tank is connected the subassembly is placed to the membrane, the other end that the recess was kept away from to the liquid hold up tank is equipped with butt portion, butt portion locates in the centrifugate holding intracavity, it butt portion extends to centrifuging tube bottom.

Optionally, the membrane placing assembly comprises a first assembly and a second assembly, the first assembly is detachably connected to the second assembly, the separation membrane is arranged between the first assembly and the second assembly, and the separation membrane is clamped to the connecting portion of the first assembly and the connecting portion of the second assembly.

Optionally, the diameter of the second component is smaller than that of the first component, the second component is accommodated in the groove of the liquid storage tank, and the diameter of the groove of the liquid storage tank is matched with that of the first component.

Optionally, the second assembly is provided with a communicating hole, and the outer side wall of the first assembly is provided with a communicating groove.

Optionally, the sample adding cylinder and the membrane placing component are made of polyamide, and the liquid storage tank is made of metal aluminum.

Optionally, the separation membrane is a polycarbonate filter membrane, the average membrane pore size of the separation membrane is 6.9 ± 0.6 μm, and the porosity of the separation membrane is 3% -4%.

Based on the same inventive concept, the embodiment of the present invention further provides a cancer cell capturing method, which uses the cancer cell capturing system as described above, and the method includes:

and injecting the material to be separated into the membrane separation assembly, and then rotating the horizontal rotor to enable the centrifugal tube to carry out horizontal rotor centrifugation, so that separation is completed, and the target cell and the centrifugate are obtained.

Optionally, the method further includes:

the capture system is pre-treated to achieve fluid-assisted separation and to prevent cell adhesion during centrifugation.

One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

the cancer cell capturing system provided by the embodiment of the invention improves the condition that a blood sample flows from the inner side to the outer side of a disc after being added on the membrane in the existing filtering method by using the design of an assembly device for centrifugal membrane filtration by using a horizontal angle rotor on the basis of the existing fluid auxiliary separation, forms continuous fluid during centrifugation, has small pressure drop on two sides of the membrane, solves the problems that the cells are stressed differently on the inner side and the outer side of the membrane during centrifugation, simultaneously, the liquid flow is always vertical to the plane of the separation membrane during centrifugation, the liquid flow is uniformly distributed, solves the problems that the edge is easy to be accumulated during centrifugation at present, the membrane filtration efficiency and the cell survival are influenced, and the like, and simultaneously solves the defects of complicated operation, high cost and the like of the prior technical route.

The foregoing description is only an overview of the technical solutions of the present invention, and the embodiments of the present invention are described below in order to make the technical means of the present invention more clearly understood and to make the above and other objects, features, and advantages of the present invention more clearly understandable.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on the drawings without creative efforts.

FIG. 1 is a schematic diagram of a system according to an embodiment of the present invention;

FIG. 2 is a schematic structural diagram of a membrane separation module provided by an embodiment of the present invention;

FIG. 3 is a flow chart of a method provided by an embodiment of the present invention;

FIG. 4 is a graph of the capture efficiency of HeLa cells at different densities in blood provided by an embodiment of the present invention;

FIG. 5 is a graph of HeLa, SW620, MDA-MB-231 and H226 cell capture efficiency in blood provided by an embodiment of the present invention;

FIG. 6 is a fluorescence image of cancer cells and leukocytes on a membrane after filtration as provided by an embodiment of the invention;

FIG. 7 is a graph showing the efficiency of capturing Hela cells in blood and the leukocyte removal rate of Hela cells at different PBS washing times after centrifugation as provided in the examples of the present invention;

FIG. 8 shows the capture efficiency of H226 cells at different densities in a medium provided by an example of the present invention;

FIG. 9 is a graph of HeLa, SW620, MDA-MB-231, and H226 cell capture efficiency in media provided by examples of the present invention;

FIG. 10 is a fluorescence image of cancer cells and leukocytes collected by the filtered outflow device provided by embodiments of the invention;

FIG. 11 is a graph showing the efficiency of capturing Hela cells and the effect of removing leukocytes under conditions of a fixed rotor and non-rapid centrifugation according to an embodiment of the present invention;

FIG. 12 is a graph of the viable staining of cells on days 1 and 10 for the experimental and control groups provided by the examples of the present invention;

FIG. 13 is a statistical graph of the survival rates of cells from experimental and control groups provided by an example of the present invention;

FIG. 14 is a bright field plot of cells grown over time for 1-5 days in experimental and control groups as provided by an example of the present invention;

FIG. 15 is a graph showing the growth of cells in the experimental group and the control group according to the example of the present invention;

reference numerals: 1-centrifuge tube, 11-centrifuge tube body, 12-membrane separation component, 121-membrane placement component, 121 a-first component, 121 b-second component, 122-separation membrane, 123-sample adding barrel, 124-liquid storage tank, 124 a-groove, 124 b-abutting part, 2-horizontal rotor, 21-rotating part and 22-hanging part.

Detailed Description

The present invention will be specifically explained below in conjunction with specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly presented thereby. It will be understood by those skilled in the art that these specific embodiments and examples are illustrative of the invention and are not to be construed as limiting the invention.

Throughout the specification, unless otherwise specifically noted, terms used herein should be understood as having meanings as commonly used in the art. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is a conflict, the present specification will control.

Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or can be prepared by existing methods.

In order to solve the technical problems, the general idea of the embodiment of the application is as follows:

the applicant finds in the course of the invention that: the cancer cell capturing method based on centrifugal membrane filtration has the advantages of simplicity, rapidness, high flux and the like, but the problems of blockage, high pressure drop and cell viability impairment exist generally, a plurality of researches improve a filtration membrane or a filtration method, but a space for improving the capturing efficiency of cells, the survival of cells, the convenience of manufacturing and using devices and the universality of application range exists, and a simple, rapid, high-efficiency, high-flux and wide-applicability CTC capturing method is still needed in the face of a large-volume complex clinical liquid sample.

According to an exemplary embodiment of the present invention, there is provided a capturing system of cancer cells, the system including: centrifuge tubes and horizontal rotors.

The centrifugal tube comprises a centrifugal tube body and a membrane separation assembly, the centrifugal tube body is provided with a hanging piece, an assembly installation cavity and a centrifugate containing cavity are formed in the inner cavity of the centrifugal tube, the centrifugate containing cavity is formed in one end far away from the hanging piece, and the membrane separation assembly is installed in the assembly installation cavity;

generally, a 50mL centrifuge tube is typically used for the centrifuge tube body. In other embodiments, the specification of the centrifuge tube can be adjusted by those skilled in the art according to actual needs, including but not limited to 5mL, 15mL, 250mL, and the like.

In some embodiments, the membrane separation assembly is mounted into a 50mL centrifuge tube to achieve fluid assisted membrane filtration with a conventional centrifuge, while the use of horizontal rotor centrifugation ameliorates the problem of uneven cell stress on the membrane, resulting in uniform distribution of cells on the membrane during filtration, resulting in simple, fast, and efficient CTC capture.

In some embodiments, a membrane separation module comprises: subassembly, separation membrane and a sample adding section of thick bamboo are placed to the membrane, the separation membrane is located in the subassembly is placed to the membrane, add a sample section of thick bamboo connect in subassembly one end is placed to the membrane. Typically, the sample addition cartridge is also removably attached to one end of the membrane placement assembly, typically by a threaded connection, snap fit, or the like.

In this example, the sample addition cylinder is cylindrical and adapted to a 50mL centrifuge tube, the outer diameter of the sample addition cylinder is 23.5mm, the height of the sample addition cylinder is 70mm,

further, the membrane placing assembly comprises a first assembly and a second assembly, wherein the first assembly is detachably connected with the second assembly, and the detachable connection mode comprises the following steps: joint, threaded connection, bolt connection etc, the release film is located between first subassembly and the second subassembly, just the release film joint in the connecting portion of first subassembly and second subassembly.

Furthermore, the diameter of the second assembly is smaller than that of the first assembly, the second assembly is contained in the groove of the liquid storage tank, and the diameter of the groove of the liquid storage tank is matched with that of the first assembly.

The connection of the first assembly and the second assembly is exemplified by clamping, the inner side of the bottom of the first assembly is provided with a protruding part, the outer side of the top of the second assembly is also provided with a protruding part which is mutually matched, when the separation membrane is installed, the separation membrane is randomly laid on the connecting end of the first assembly or the second assembly, then the first assembly and the second assembly are clamped, and meanwhile, the installation of the separation membrane is realized.

Specifically, to accommodate the size of the applicator, the diameter of the first member is 24.5mm and the diameter of the second member is 20mm in this embodiment.

The material of the sample adding cylinder and the membrane placing component can be any material, in this embodiment, the material of the membrane placing component is preferably polyamide, and in other embodiments, a person skilled in the art can select other materials to prepare the sample adding cylinder and the membrane placing component as required; in this embodiment, the material of the liquid storage tank is preferably metal, specifically, aluminum, and in other embodiments, a person skilled in the art may select other materials to prepare the liquid storage tank as needed. All the parts can be manufactured by turning and other processes, the operation is simple and convenient, and the parts can be repeatedly used.

In some embodiments, the membrane separation assembly further comprises: the liquid hold up tank, the recess end of liquid hold up tank is connected the subassembly is placed to the membrane, the other end that the recess was kept away from to the liquid hold up tank is equipped with butt portion, butt portion locates in the centrifugate holding intracavity, it butt portion extends to centrifuging tube bottom.

In an embodiment, to accommodate the size of the membrane placement module, the inner diameter of the groove of the fluid reservoir is 24.8mm, and the lower fluid reservoir can be placed directly outside the first module.

The second component is provided with a communicating hole for facilitating the liquid after centrifugation to reach the bottom of the centrifuge tube body, and the outer side wall of the first component is provided with a communicating groove. In this embodiment, the first assembly has a plurality of shallow thin grooves on its outer side and a plurality of semicircular notches on its bottom.

In some embodiments, the system can be used for separating cancer cells, when the cancer cells are separated, the separation membrane is a polycarbonate filter membrane, the average membrane pore size of the separation membrane is 6.9 +/-0.6 microns, and the porosity of the separation membrane is 3% -4%. Specifically, the separation membrane was a commercial Isopore polycarbonate 8 μm filter membrane, and the average membrane pore size was 6.9 ± 0.6 μm (n ═ 100) and the porosity was 3.3% as measured by Scanning Electron Microscopy (SEM) and other filters of different pore distribution and porosity were adjusted to the appropriate size and placed in the device. The membrane has no liquid residue after blood filtration, which indicates that the device can rapidly and efficiently process blood samples, and it should be noted that, in other embodiments, a person skilled in the art can adjust the selection of the membrane according to actual needs, and only the requirement that the pore diameter of the membrane is smaller than the diameter of the particles to be separated is satisfied.

The horizontal rotor comprises a rotating part and a hanging part, the rotating part is rotatably arranged on the working platform, and the hanging part is connected to the rotating part; the hanging part is used for hanging the hanging part so as to realize centrifugal separation of the centrifugal tube.

On the basis of the existing fluid auxiliary separation, the design of an assembly device for centrifugal membrane filtration by using a horizontal angle rotor improves the condition that a blood sample flows from the inner side to the outer side of a disc after being added on the membrane in the existing method for filtration, and solves the problems that cells are easily accumulated on the edge to cause blockage and influence the membrane filtration efficiency and the cell survival because the cells are stressed at different sizes on the inner side and the outer side of the membrane in the centrifugation.

In this embodiment, the sample application cartridge can apply 15mL of sample solution at most, and the membrane filtration centrifugation is usually performed for 1min, so that the rapid separation of a large volume of liquid sample can be realized. The whole system can be independently used for quickly detecting cancer cells, and can also be combined with other cancer cell capturing devices to be used as a primary screening device.

According to another exemplary embodiment of the present invention, there is provided a method for capturing cancer cells, using the system for capturing cancer cells as described above, the method including:

s1, preprocessing a capture system to realize fluid-assisted separation and prevent cell adhesion in a centrifugal separation process;

s2, injecting a material to be separated into the membrane separation assembly, then rotating the horizontal rotor to enable the centrifugal tube to carry out horizontal rotor centrifugation, and completing separation to obtain target cells and centrifugal liquid.

Specifically, the filter membrane was cut to an appropriate size before using the device, the device was assembled into the device and then placed in a 50mL centrifuge tube, 3mL of 1% serum albumin (BSA) was added to the cylindrical sample addition chamber in order to achieve fluid-assisted separation during centrifugation and prevent cell adhesion during centrifugation, and the cylindrical sample addition chamber was centrifuged at 14g Relative Centrifugal Force (RCF) (300rpm) for 1min and then left at room temperature for 30 min. To capture cancer cells, 1mL of cell suspension or cancer cell-contaminated blood was added to the device, centrifuged at 14g RCF (300rpm) for 1min, filtered, and then centrifuged under the same conditions with 1mL of PBS if washed with PBS. After centrifugation, the device is disassembled, the filter membrane is taken out and placed in a 6-well plate, liquid in a liquid storage tank is transferred into a 50mL centrifuge tube, 1mL PBS is added to wash the liquid storage tank and transfer the liquid again, the solution in the centrifuge tube is centrifuged for 5min under the condition of 225g RCF (1200rpm), part of supernatant is discarded, mixed and transferred into a 96-well plate, a fluorescence microscope and an Andor EMCCD camera are used for imaging and observing cells on the membrane in the 6-well plate and in the 96-well plate, and the number of different cells is counted.

It should be noted that the above is only illustrated for the separation of cancer cells, and in other embodiments, the systems and methods provided above can be applied to the separation of other particles as well.

The cancer cell capturing system and method of the present application will be described in detail below with reference to examples, comparative examples, and experimental data.

Staining of cancer cells of cell lines: the cell culture dish in the incubator is taken out, the culture medium is sucked out, 1mL of PBS is added for cleaning, the PBS is discarded, 1mL of trypsin is added, and the cell culture dish is placed in the incubator for digestion for 5 min. After cell digestion, 2mL of the medium was added, mixed well and transferred to a centrifuge tube, centrifuged for 5min at 225g RCF (1200rpm), and then added with PBS and centrifuged and washed 1 time. The preparation of CellTracker Orange CMRA dye solution is carried out in a lightproof super clean bench, 5 mu L of dye solution is added into 500 mu L of PBS, the prepared dye solution is added after the cells are washed, and the cells are wrapped by tinfoil paper and put into an incubator for incubation for 30 min. After staining, the cells were washed 1 time by centrifugation with PBS. 10 μ L of stained cancer cells were counted using a hemocytometer to determine cell density and added to the medium or blood by gradient dilution to a final density of 200-500 cells/mL.

Cell capture at different densities and different cell lines: to assess the capture efficiency of cells at different densities, cancer cells were added to the culture medium or rabbit blood at different concentrations. For the capture efficiency of cancer cells of different cell lines, CTC capture experiments were performed using cells of four cell lines, HeLa, SW620, MDA-MB-231, and NCI-H226. The number of cells on the membrane and those flowing out were counted under a microscope after centrifugation, and the capturing efficiency of cancer cells was calculated by the number of cancer cells on the membrane/(the number of cancer cells on the membrane + the number of cancer cells flowing out) × 100%.

Leukocyte staining in blood: the rabbit blood in the blood collection tube was centrifuged for 5min under 225g RCF (1200rpm), white cell nuclei in the blood were stained with Hochest 33342 stain in a dark super clean bench, 5. mu.L of the stain was added to 1mL of PBS, the blood cells were centrifuged, the prepared stain was added, wrapped with tinfoil paper and placed in an incubator for 40 min. After staining, cells were washed 1 time with PBS and blood was diluted 10-fold with medium for cancer cell doping experiments. The removal efficiency of White Blood Cells (WBCs) was calculated by the log of the number of white blood cells removed, which is equal to log (total white blood cells/number of white blood cells on the membrane).

The device uses: the filter membrane was cut to the appropriate size and placed in the device, the device was placed in a 50mL centrifuge tube, and 3mL of 1% serum albumin (BSA) was added to the loading chamber for fluid assisted separation and to prevent cell adhesion during centrifugation, and after centrifugation for 1min at 14g Relative Centrifugal Force (RCF) (300rpm), the chamber was left at room temperature for 30min (non FAST centrifugation without this loading step). For the experiment, 1mL of cell suspension or cancer cell-contaminated blood was added to the device, centrifuged for 1min at 14g RCF (300rpm, horizontal rotor centrifuge or angle rotor centrifuge), filtered, and centrifuged under the same conditions with 1mL of PBS if washed with PBS. And taking out the filter membrane after centrifugation, putting the filter membrane into a 6-well plate, transferring the liquid in the liquid storage tank and the liquid in the liquid storage tank into a 50mL centrifuge tube, adding 1mL PBS to wash the liquid storage tank, transferring the liquid again, centrifuging the solution in the centrifuge tube for 5min under the condition of 225g RCF (1200rpm), transferring the solution into a 96-well plate, performing imaging observation on the cells on the membrane in the 6-well plate and in the 96-well plate by using a fluorescence microscope and an Andor EMCCD camera, and counting the number of different cells.

Cell collection after centrifugation: MDA-MB-231 cells are digested, centrifuged, adjusted to a final density of 4000-10000 cells/mL in a culture medium, centrifuged and filtered in a device, then washed for 2 times by adding 1mL PBS, the device is taken out, a reservoir at the bottom is removed, the device is reversely placed in a new 50mL centrifuge tube, 1mL of the culture medium is added on a membrane, centrifuged for 10min under the condition of 978g RCF (2500rpm) and then repeatedly centrifuged for 1min by adding the culture medium, the solution in the tube is transferred to a 96-well plate after centrifugation, and 100 mu L of the cells which are not centrifuged and filtered in each well are taken as a control group and put into the 96-well plate.

Cell survival assay: putting cells in a 96-well plate in an incubator, respectively using a Calcein-AM/PI staining kit to determine the survival condition of the cells after 4-8h and on the 10 th day, adding 1 mu L Calcein-AM and 0.2 mu L PI into 10 mu L buffer solution in the kit, uniformly mixing, adding 1mL PBS to prepare a staining solution, adding 200 mu L staining solution into each hole after the culture medium of the cells in the 96-well plate is sucked out, incubating for 20min in the incubator, photographing the cells under a microscope after staining, and respectively displaying green fluorescence and red fluorescence by live cells and dead cells.

Cell growth curve determination: the captured cancer cells were treated at 37 ℃ with 5% CO₂The culture box is used for culturing and growing, cells are taken out on days 1, 2, 3, 4 and 5 respectively, the cells are photographed under a microscope bright field, the number of the cells is counted, growth curves are drawn according to the number of the cells of an experimental group and a control group respectively, and the cells are fitted according to a cell growth formula.

As shown in FIG. 4, which is a graph of the capture efficiency of HeLa cells with different densities in blood, it can be obtained from the graph that when the cell density is in the range of 100-600 cells/mL, the captured number has a better linear relationship, and the average capture rate is 95.1%;

as shown in fig. 5, which is a graph of the cell capturing efficiency of HeLa, SW620, MDA-MB-231 and H226 in blood (n ═ 3), the capturing efficiency of the four cell lines was 94.6%, 97.8%, 95.7% and 94.9%, respectively;

as shown in fig. 6, which is a fluorescence image of cancer cells and leukocytes on the membrane after filtration, red color indicates cancer cells, and blue color indicates leukocytes, and most of the cancer cells and a small amount of leukocytes are captured on the membrane;

as shown in fig. 7, which is a graph of the capturing efficiency of Hela cells in blood and the leukocyte removal rate (n ═ 3) at different PBS washing times after centrifugation, the leukocyte removal rate was positively correlated with the number of membrane washes after filtration, the removal rate was 98.79% to 99.72% at 0-4 washes, and the capturing efficiency of cancer cells was 90% or more at different washing times;

as shown in FIG. 8, for the capture efficiency of H226 cells with different densities in the culture medium, it can be obtained from the figure that when 100-600/mL cells with different densities are added, the captured number of the cells shows a good linear relationship, and the capture rate is about 95.2%;

as shown in fig. 9, which is a graph of cell capture efficiency of HeLa, SW620, MDA-MB-231 and H226 in the medium (n ═ 3), the cell capture efficiency of the four cell lines was 96.5%, 95.0%, 95.5% and 95.4%, respectively.

FIG. 10 is a fluorescence view of cancer cells and leukocytes collected by the filtration and elution device, in which red represents cancer cells and blue represents leukocytes, and from which a very small number of cancer cells and the vast majority of leukocytes are eluted;

as shown in fig. 11, which is a graph of the capturing efficiency and the leukocyte removal effect of Hela cells under the conditions of the fixed rotor and the non-rapid centrifugation (n ═ 3), the centrifugal membrane filtration using the horizontal rotor and the fluid assistance performed better than the angular rotor and the non-FAST centrifugation, and the cancer cell capturing efficiency and the leukocyte removal efficiency were improved by 1.7% and 9.5%, 6.8% and 15.3%, respectively;

the isolated cancer cells were subjected to viability and growth tests, resulting in FIGS. 12-15.

As shown in fig. 12, the survival staining patterns of the cells of the experimental group and the control group on day 1 and day 10 are shown, the scale bar of the survival staining patterns is 100 μm, as shown in fig. 13, the survival rate statistics (n ═ 3) of the cells of the experimental group and the control group are shown, as shown in fig. 14, the illumination field patterns of the cells of the experimental group and the control group over time are shown, the scale bar of the survival field patterns is 100 μm, as shown in fig. 15, the growth curve (n ═ 3) of the cells of the experimental group and the control group are shown, the survival rate of the cancer cells after filtration is good, and the survival rates of the cells of the experimental group and the control group on day 1 and day 10 are respectively 90.2% and 94.4%, 96.8% and 97.7%, and the survival rates of the cells of the two groups are not obviously different; after filtration, the cells of the experimental group and the control group grow well on days 1 to 5, and the fitted curves of the cell number all accord with the growth curve model of the cells.

One or more technical solutions in the embodiments of the present invention at least have the following technical effects or advantages:

(1) the system provided by the embodiment of the invention is a device for performing CTC (CTC capture) by using a horizontal rotor and fluid assistance, and solves the problems of uneven cell distribution and easy accumulation and blockage in centrifugal membrane filtration;

(2) the system provided by the embodiment of the invention adopts the horizontal rotor, and the uniform distribution of cells on the membrane can be realized during centrifugation;

(3) the method provided by the embodiment of the invention has the capture efficiencies of 96.5%, 95.0%, 95.5%, 95.4% and 94.6%, 97.8%, 95.7% and 94.9% respectively when the four cell lines of HeLa, SW620, MD-MBA-231 and NCI-H226 are captured in the culture medium and the blood, which indicates that the device can be applied to the separation of different types of cancer cells.

The removal rate of the white blood cells is 98.8%, 99.0%, 99.3%, 99.5% and 99.7% when the cells are washed 0-4 times, and the capture efficiency of the cancer cells is 94.6%, 94.4%, 96.0%, 93.1% and 92.7%.

The experimental group and the control group survived well after the centrifugal filtration on the 1 st day and the 10 th day, the survival rates are respectively 90.2 percent and 94.4 percent, 96.8 percent and 97.7 percent, the cells of the experimental group and the control group grow well after the centrifugal filtration on the 1 st to 5 th days, and the fitting curve accords with the growth curve rule model of the cells.

In terms of cancer cell capture, the use of angle rotor centrifugation in this technique was 1.7% higher than horizontal rotors, and 9.5% higher than non-FAST centrifugation. In the aspect of leukocyte removal, the removal rate is improved by 6.8% when the horizontal rotor centrifugation is used and is improved by 15.3% under the FAST condition compared with the non-FAST condition.

Finally, it should also be noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.

While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.

It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.

Claims (8)

1. A particulate matter capture system, the system comprising:

the centrifuging tube, the centrifuging tube includes centrifuging tube body and membrane separation subassembly, the centrifuging tube body is equipped with links, the centrifuging tube inner chamber is equipped with subassembly installation cavity and centrifugate holding chamber, centrifugate holding chamber is located and is kept away from link one end, the membrane separation unit mount in the subassembly installation cavity.

2. The particulate matter capture system of claim 1, wherein the membrane separation assembly comprises: subassembly, separation membrane and a sample adding section of thick bamboo are placed to the membrane, the separation membrane is located in the subassembly is placed to the membrane, add a sample section of thick bamboo connect in subassembly one end is placed to the membrane.

3. The particulate matter capture system of claim 2, wherein the membrane separation module further comprises: the liquid hold up tank, the recess end of liquid hold up tank is connected the subassembly is placed to the membrane, the other end that the recess was kept away from to the liquid hold up tank is equipped with butt portion, butt portion locates in the centrifugate holding intracavity, it butt portion extends to centrifuging tube bottom.

4. The particulate matter capture system of claim 3, wherein the membrane placement assembly comprises a first assembly and a second assembly, the first assembly is removably attached to the second assembly, the separation membrane is disposed between the first assembly and the second assembly, and the separation membrane is snapped into or adhered to a connection of the first assembly and the second assembly.

5. The particulate matter trapping system of claim 4, wherein the second member has a diameter smaller than a diameter of the first member, the second member being received in a recess of the liquid storage tank, the recess having a diameter matching the diameter of the first member.

6. The particulate matter trapping system according to claim 5, wherein the second member is provided with a communication hole, and the first member has an outer side wall provided with a communication groove.

7. The particulate matter trapping system of claim 3, wherein the cartridge and the membrane placement assembly are made of polyamide; the liquid storage tank is made of metal aluminum.

8. The particulate matter trapping system of claim 2, wherein the separation membrane has a pore size smaller than a diameter of the particles to be separated.

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