WO2022062934A1

WO2022062934A1 - Microfluidic chip-based circulating tumor/fusion cell capturing device and method

Info

Publication number: WO2022062934A1
Application number: PCT/CN2021/117878
Authority: WO
Inventors: 刘宗彬
Original assignee: 深圳市瑞格生物科技有限公司
Priority date: 2020-09-22
Filing date: 2021-09-13
Publication date: 2022-03-31
Also published as: CN213624081U

Abstract

Disclosed are a microfluidic chip-based circulating tumor/fusion cell capture device and method. The capture device comprises a microfluidic chip used to perform cell separation and capture of a sample, and a control system in sealed communication with the microfluidic chip so as to provide a fluid driving force to the microfluidic chip for completing cell separation and capture. The control system comprises an air source device, an air pressure adjustment device, and a human-computer interaction device.

Description

Circulating tumor/fused cell capture device and method based on microfluidic chip

technical field

The present invention relates to a circulating tumor/fused cell capture device based on a microfluidic chip and a method thereof, in particular to a circulating tumor/fused cell capture device driven by pneumatic pressure to control fluid and its method application in cell sorting .

Background technique

At present, the continuous rise of tumor morbidity and mortality has become one of the serious problems in today's society. At this stage, the discovery and diagnosis of most tumors still rely on imaging examinations, traditional tumor markers and tissue biopsy. Traditional invasive tissue biopsy needs to remove a tissue or cell sample from the patient. This method often brings patients with Comes with a certain risk and pain. More importantly, tissue biopsy is not suitable for repeated extraction and is prone to complications, and it has the risk of spreading cancer cells.

With the development of science and technology, "liquid biopsy", which analyzes tumor information by drawing a small amount of blood, has become an important branch in the field of tumor and testing. With its advantages of non-invasiveness, convenience, and real-time performance, it gradually has the trend of catching up and replacing traditional biopsy. . Biomarkers of liquid biopsy mainly include tumor cells, DNA fragments and exosomes released by tumor tissue into the blood. Among them, Circulating Tumor Cell (CTC) is a general term for various types of tumor cells existing in peripheral blood. It is an important cause of tumor metastasis and an important detection object of liquid biopsy. Due to the very small number of CTCs in patient blood samples, there are only 1-10 CTCs per milliliter of whole blood in patients with tumor metastases, making CTC isolation and detection very challenging. At present, CTC enrichment methods include biochemical characteristic enrichment method and physical characteristic enrichment method. The heterogeneity of CTC in physical properties cannot be overcome by the membrane filtration method, and the membrane filtration method is also prone to clogging and low separation flux. CTC technology based on microfluidic chips can not only detect the number of CTCs, but also separate live CTCs for subsequent research and analysis, and will be the mainstream technology for CTC detection in the future.

Circulating fusion cells Fusion Cell, CFC) is a new type of cell discovered in the process of CTC separation and detection. It is a fusion of tumor cells and immune cells (macrophages, B cells, T cells, neutrophils, etc.) Mononuclear or multinucleated structure, which can express both tumor cell and immune cell markers, in a variety of known solid tumors (such as lung cancer, liver cancer, neuroblastoma, Ewing sarcoma, gastric cancer, colon cancer, breast cancer, thyroid cancer etc.), and the number of fused cells is generally higher than that of CTCs, which may be related to the immune escape of tumors. Therefore, the combination of CTCs and fused cells can be of great importance for early diagnosis and screening of tumors, curative effect monitoring, and prognosis evaluation. significance.

technical solutions

In order to solve the current problem of difficulty in separating and capturing CTCs and fused cells, the present invention provides a circulating tumor/fused cell capture device based on a microfluidic chip and a method thereof, which can not only efficiently, simply and efficiently separate and capture CTCs and fused cells. At the same time, it can also separate and capture target cells in various biological samples such as blood, body fluids, and bone marrow. The technical scheme of the device is as follows:

A circulating tumor/fused cell capture device based on a microfluidic chip, comprising an operating table, a microfluidic chip for separating and capturing samples from a sample, and a microfluidic chip arranged on the operating table and the microfluidic chip. A container used in conjunction with a flow control chip for loading a sample or solution, the container can be a test tube, and a test tube rack for placing the test tube can be provided on the operating table; the capture device also includes a pair of microfluidic chips. A control system for providing fluid driving force to complete the separation and capture of cells, the control system includes an air source device, an air pressure regulating device connected with the air source device for controlling the output air pressure of the air source device, and an air pressure adjustment device for setting and a human-computer interaction device for displaying the output air pressure; the human-computer interaction device is connected to the air pressure regulating device through a circuit control device; the air pressure regulating device is sealed and connected to the The container (such as a test tube) provided with precise and stable air pressure is used to drive the sample and solution to flow in the microfluidic chip to complete the separation and capture of target cells.

Further, the container (such as a test tube) includes a sample tube for providing the sample to be separated to the microfluidic chip, and a first waste for collecting the main waste liquid after the primary separation of the microfluidic chip. Liquid collection tube, a solution tube for providing secondary separation solution and fluid power to the sample after primary separation to remove the main waste liquid, and a second solution tube for collecting secondary waste liquid after secondary separation by microfluidic chip Waste liquid collection tube and enrichment liquid collection tube for collecting the target cell enrichment liquid obtained after secondary separation of the microfluidic chip, the sample tube, the first waste liquid collection tube, the solution tube, the second waste liquid collection tube The liquid collection tube and the enriched liquid collection tube are respectively sealed and connected through a thin conduit (such as a thin PTFE tube) and the corresponding inlet and outlet on the described microfluidic chip.

Further, the microfluidic chip is respectively provided with a sample inflow port corresponding to the sample tube, a first waste liquid outflow port corresponding to the first waste liquid collection tube, and a solution inflow port corresponding to the solution tube. , a second waste liquid outflow port corresponding to the second waste liquid collection pipe, and an enriched liquid outflow port corresponding to the enriched liquid collection pipe.

Further, the air source device can be one or more micro air pumps, or an external air pump, or an external air cylinder in the control system, the air pressure regulating device can be an electric proportional valve, and the circuit control The device can be a PLC (Programmable Logic Controller) or a self-developed microcontroller.

Further, one side of the operating table is provided with 5 tracheal interface devices connected from the air pressure regulating device, one end is respectively connected to the air pressure output ports of the 5 air pressure regulating devices in the control system, and the other end is sealed and connected to the sample tube and the first waste liquid respectively. The collection tube, the solution tube, the second waste liquid collection tube and the enriched liquid collection tube; the number of trachea interface devices can be increased or decreased according to the separation needs; the operating table is provided with a transparent protective cover shell.

Further, the adjustment and display of the air pressure are realized by the human-computer interaction device through the circuit control device, and the human-computer interaction device includes a touch screen which can be interacted with the human machine; the touch screen can input and output the air pressure adjustment device to each For the air pressure setting value and actual air pressure output value of the pipeline, the user can set the required air pressure value through the touch screen, and the circuit control device outputs the corresponding air pressure by controlling the air pressure adjustment device to achieve precise control; the air pressure adjustment device has a calibration function; The air pressure display unit can be mBar.

Further, a sealing cover with an opening is provided at the top opening of the container (such as a test tube), and the opening on the sealing cover includes a gas connection between the container (such as a test tube) and the outlet of the tracheal interface device on the operating table. The opening of the first trachea of the circuit, and the opening of the second thin pipe for connecting the liquid circuit between the container (such as a test tube) and the microfluidic chip, thereby forming a fluid system of a sealed liquid circuit driven by air pressure.

Further, the microfluidic chip is composed of a base layer and a structural layer, the base layer is made of glass or polymer, the structural layer is generally prepared from a polymer, and the polymer layer can be made of polydimethylsiloxane. alkane (PDMS), the structural layer can be one or more layers; each layer of the chip is bonded and pasted after plasma surface activation; the structural layer is provided with a series of enrichment targets based on hydrodynamics and spatial deformation The composite micro-pillar array structure of cells, the internal structural layer is connected with the surface structural layer through channel collection and perforation, and multiple structural layers are conducive to the arrangement of the micro-pillar array of the extended chip on the limited surface and the use of fluid channels. interlayer collection. In an embodiment of the present invention, the chip is composed of a glass base layer and two upper and lower polymer layers (ie, upper and lower structural layers), and a series of lateral displacements with certain lateral displacements arranged according to critical dimensions are arranged on the polymer layers. (Deterministic Lateral Displacement) the structure of the composite micro-pillar array, the blood samples are separated between the micro-pillar arrays; the upper structural layer is the guide layer, and the lower structural layer is the separation layer; the guide layer is provided with an inlet and outlet, including a general first Waste liquid outlet, the first waste liquid outlet is connected to a plurality of separate (6 in one embodiment) main waste liquid outlets on the separation layer in the form of a series of micro-column arrays, and the blood is separated and enriched by the separation layer After that, the main waste liquid is collected through the first waste liquid outlet on the diversion layer and then flows out, thereby reducing the number of waste liquid outlets; a series of waste liquid outlets for extending the second waste liquid outlet and rich liquid outlet are also arranged on the diversion layer. For the micro-column array of the liquid collection outlet, by setting corresponding outlets at different extension positions, different fluid path lengths are formed so that the ratio of the amount of cells in the second waste liquid outlet and the enrichment liquid outlet can be changed.

Further, the application of the above-mentioned microfluidic chip-based circulating tumor/fused cell capture device includes but is not limited to any of the following: (1) separation and/or capture of circulating tumor cells, circulating tumor cells in peripheral blood samples Cell clusters, fused cells; (2) isolation and/or capture of tumor cells, tumor cell clusters, fused cells in pleural effusion, ascites, lymph, urine or bone marrow samples; (3) isolation and/or capture Nucleated red blood cells in peripheral blood or cord blood samples; (4) isolation and/or capture of circulating endothelial cells in peripheral blood samples; (5) isolation and/or capture of peripheral blood, cord blood, pleural effusion, ascites , leukocytes, T cells, B cells, lymphocytes, monocytes, granulocytes, natural killer cells, dendritic cells, macrophages or hematopoietic stem cells in urine, cerebrospinal fluid or bone marrow samples; (6) isolation and/or Or capture red blood cells or platelets in peripheral blood, umbilical cord blood, pleural effusion, ascites effusion, urine or bone marrow samples; (7) Separate and/or capture peripheral blood, pleural effusion, ascites effusion, urine, saliva , Bacteria or viruses in plasma, serum, cerebrospinal fluid, semen, prostatic fluid or vaginal secretion samples; (8) isolation and/or capture of sperm in semen samples.

Further, the specific capture method of the above-mentioned microfluidic chip-based circulating tumor/fused cell capture device includes the following steps:

(1) The blood sample to be separated is contained in the sample tube. The first waste liquid collection tube contains the rinsing solution for rinsing the chip and the catheter. The solution tube contains the solution for rinsing and secondary separation of the sample. The waste liquid collection tube and the enrichment liquid tube are empty tubes without prefilled solution. Each test tube is connected to the 5 tracheal ports on one side (such as the rear side) of the operating table through the first tracheal opening on the top sealing cover, and a thin tube (such as a thin PTFE tube) is inserted through the second tube opening on the sealing cover. , one end of the thin tube is connected to the corresponding fluid inlet and outlet on the chip (the sample inflow port corresponding to the sample tube, the first waste liquid outflow port corresponding to the first waste liquid collection tube, the solution inflow port corresponding to the solution tube, and the first waste liquid outflow port corresponding to the first waste liquid collection tube. The second waste liquid outflow port corresponding to the second waste liquid collection pipe and the enrichment liquid outflow port corresponding to the enrichment liquid collection pipe), and the other end is inserted into the bottom of the test tube (wherein the second waste liquid collection pipe and the enrichment liquid pipe are not pre- solution without inserting into the bottom), thus forming a hermetically air-driven fluidic separation system between each container (such as a test tube) and the chip.

(2) Set the air pressure of the first waste liquid collection tube, the sample tube, the solution tube, the second waste liquid collection tube and the enriched liquid collection tube at a certain time interval in turn, so that the rinsing in the first waste liquid collection tube The solution and the solution in the solution tube flow through the whole chip and pipeline, and play the role of rinsing and emptying the bubbles in the chip and pipeline.

(3) After the bubbles are discharged, set the air pressure of the sample tube, the first waste liquid collection tube, the solution tube, the second waste liquid collection tube and the enrichment liquid collection tube at the same time, so that the blood sample flows in from the chip inlet, buffering The liquid flows in from the solution inlet, the main waste blood produced after the primary separation flows out from the first waste liquid outlet, the secondary waste blood produced after the secondary separation flows out from the second waste liquid outlet, and the enriched liquid flows out of the enriched liquid outlet outflow. During the separation process, the air pressure parameters of the separation system can be adjusted at any time as needed, so that the separation time and capture efficiency can be changed: by increasing or decreasing the air pressure of the sample tube, the flow rate of the sample entering the chip can be increased or decreased accordingly. In addition, the first waste liquid collection tube is used for the rinsing of chips and pipelines in the early stage and the collection of main waste liquid in the later stage. The ratio of the first waste liquid collection tube to the amount of cells entering the secondary separation can be adjusted by slightly increasing or decreasing the air pressure of the first waste liquid collection tube, so that the cells entering the second waste liquid collection tube and the cells in the enrichment tube are The amount of separation is increased or decreased accordingly, and the desired amount of target cells can be flexibly adjusted.

(4) After the separation is completed, increase the air pressure of the solution pipeline, and adjust the air pressure of the other pipelines to 0, so that the solution in the solution tube fills the chip and all the thin tubes to prevent the pollution caused by the residual waste blood. Finally, the pressure of the solution pipeline is also adjusted to 0, and the shutdown is completed after the end.

In one embodiment, the sample tube is filled with 6 mL of diluted blood sample, the first waste liquid collection tube is filled with 5 mL of rinsing solution, the solution tube is filled with 20 mL of secondary separation solution, and the second waste liquid is filled with 20 mL of secondary separation solution. The collection tube and the enrichment collection tube are not filled with solution.

After turning on the instrument, the first waste liquid collection tube first set the air pressure to 430 mBar, and maintained for 40 s; then set the sample tube air pressure to 420 mBar, and kept the original pressure value in the first waste liquid collection tube unchanged, and continued to maintain it for 30 s; Then set the air pressure of the solution tube to 450 mBar, and keep the air pressure of the first two test tubes unchanged for 30 s; finally set the pressure of the second waste liquid collection tube and the enriched liquid collection tube to 250 mBar, and keep the first three test tubes. The air pressure remains unchanged for another 3 min, at which time the chip and pipeline have been rinsed and the air bubbles have been emptied.

Then set the air pressure of the second waste liquid collection tube and the enriched liquid collection tube to 0 mBar, the solution tube air pressure to 370 mBar, the first waste liquid collection tube air pressure to 388 mBar, and the sample tube air pressure to 520 mBar. The sample is officially separated. During the separation process, the amount of cells flowing into the enriched liquid collection pipe can be adjusted by fine-tuning the value of the air pressure of the first waste liquid collection pipe. If you want to collect a larger amount of cells in the enrichment solution, then slightly increase the air pressure of the first waste liquid collection tube; air pressure.

After the separation is completed, set the pressure of the sample tube and the first waste liquid collection tube to 0 mBar, and set the pressure of the solution tube to 800 mBar. After the solution in the solution tube is filled with the chip and the thin tube, set the pressure of the solution tube to 0 mBar, and complete the shutdown.

beneficial effect

The beneficial effect of the above-mentioned cell capture method based on microfluidic chip is that whole blood is directly loaded and separated in one step, without multi-step separation operation, and without the participation of adsorption methods such as magnetic beads and antibodies, and the separation time is short, and most samples are separated within 20 minutes. The separation can be completed within 30 minutes, with high separation efficiency, high cell activity, and low manufacturing cost. As a microfluidic chip sorting cell platform, it has simple operation, stable performance and strong practicability. The blood sample and buffer solution enter the microfluidic chip at a uniform and stable flow rate through the precise air pressure driving force of the device. After the physical separation of the micro-pillar array structure inside the chip, the suspension containing the target cells flows out from the enrichment solution collection tube , the main waste liquid and the secondary waste liquid flow out from the first waste liquid collection pipe and the second waste liquid collection pipe respectively. The resulting target cell suspension has high separation activity, except for immunofluorescence and FISH Staining can also be applied to downstream digital PCR, single-cell gene sequencing, cell culture, drug sensitivity testing and other analyses to provide precise diagnosis and treatment services for tumor patients. Another advantage of the present invention is that the air pressure parameters for separating cells can be adjusted autonomously, and the separation time, cell collection amount, etc. can be flexibly adjusted according to user needs and different sample characteristics.

Description of drawings

1 is a perspective view of the device;

Fig. 2 is the schematic diagram after the transparent casing of the device is removed;

3 is a schematic diagram of the internal structure of the device;

Figure 4 is a diagram of cell identification and classification after separation.

Embodiments of the present invention

The present invention will be further explained below in conjunction with the accompanying drawings.

A circulating tumor/fused cell capture device based on a microfluidic chip, the capture device comprises an operating table (1), and a device provided on the operating table (1) for performing CTC and fusion on a sample A microfluidic chip (11) for cell separation and capture, and a test tube rack (12) for placing test tubes (01); the capture device further includes a control system (2), and a control system (2) provided in the control system (2) ), the air pressure regulating device connected with the air source device for controlling the air pressure output size and the man-machine interaction device for setting and displaying the output air pressure size, the air source shown in FIG. 3 The device is a micro air pump (21); the displayed air pressure adjusting device is an electric proportional valve (22), and the human-computer interaction device is a touch screen (23) capable of human-machine interaction, and the touch screen (23) is controlled by a circuit The device adjusts the electrical proportional valve (22), so as to control and adjust the air pressure value of each test tube (01); the air source device and the air pressure regulating device are sealedly connected to the test tube (01) through the air tube and the test tube (01) Provide air pressure to drive the sample to flow in the microfluidic chip (11) for separation; the connection method is firstly that the air source device is connected to the air pressure regulating device, and then the air pressure regulating device is connected to the test tube (01), and the test tube is then connected to the test tube (01). Connect to the microfluidic chip through a thin tube.

In one embodiment, there are five test tubes (01) in total, as shown in Figures 2 and 3, which are respectively a sample tube, A first waste liquid collection tube for collecting the main waste liquid separated by the microfluidic chip (11), a solution tube for providing secondary treatment solution and hydrodynamic force to the sample after removing the main waste liquid, for collection a second waste liquid collection tube for the secondary waste liquid separated by the microfluidic chip (11), and an enrichment liquid collection tube for collecting the target cell enrichment liquid obtained after separation by the microfluidic chip (11), The sample tube, the first waste liquid collection tube, the solution tube, the second waste liquid collection tube and the enriched liquid collection tube are sealedly connected to the microfluidic chip (11) through a thin tube. By adjusting the electrical proportional valve (22), different air pressures are given to the sample tube, the first waste liquid collection tube, the solution tube, the second waste liquid collection tube and the enriched liquid collection tube, thereby forming a pressure difference between different test tubes, prompting The flow of the blood sample in the chip; the blood sample is placed in the sample tube after simple treatment (such as dilution), and is driven by the air pressure to enter the sample inflow port of the microfluidic chip (11), and the solution in the solution tube is driven by the air pressure. The solution flow inlet of the microfluidic chip (11), the blood cells in the sample are separated and enriched by the micro-pillar array structure inside the microfluidic chip (11), the main waste liquid flows out from the outflow port of the first waste liquid collection tube, and the secondary For waste to flow out of the second waste collection tube outlet, the target cells flow out of the enrichment collection tube.

Further, the microfluidic chip (11) is respectively provided with a sample inflow port (31) connected to the sample tube, a first waste liquid outlet (35) corresponding to the first waste liquid collection tube, A solution inflow port (34) corresponding to the solution pipe, a second waste liquid outlet (33) corresponding to the second waste liquid collection pipe, and an enrichment liquid outflow outlet (32) corresponding to the enrichment liquid collection pipe. The positions of the sample tube, the first waste liquid collection tube, the solution tube, the second waste liquid collection tube and the enrichment liquid collection tube can be randomly placed in the test tube rack (12), but the connection port with the microfluidic chip (11) (31/35/34/33/32) are fixed. Figures 2 and 3 only illustrate the connection between one of the test tubes and the microfluidic chip (11). PTFE tubing).

In different embodiments, the air source device may be one or more micro air pumps built in the console, or an external air pump, or an external air cylinder.

In one embodiment, the air pressure adjusting device is an electrical proportional valve (22), as shown in Figure 3, there are five in total, which are respectively collected from the sample tube, the first waste liquid collection tube, the solution tube, and the second waste liquid The pipe and the enrichment liquid collection pipe correspond to each other through the pipeline, and there are also 5 tracheal interface devices (4) on the rear side of the operating table (1), one end is respectively connected with 5 electric proportional valves (22), and the other end is respectively connected with the sample tube , the first waste liquid collection pipe, the solution pipe, the second waste liquid collection pipe and the enriched liquid collection pipe.

In one embodiment, a transparent protective cover shell (14) is further provided on the operating table, and the transparent protective cover shell (14) can be used for the operator to observe the separation and capture process of the sample in real time.

Validation of cell separation parameters and separation time

According to the above capture method, about 5 mL of healthy human blood was placed in the sample tube, diluted to 9 mL, about 100 target tumor cells A549 stained by fluorescence were added to the blood, and different air pressure separation parameters were set (the second waste The air pressure during the separation of the liquid collection tube and the enriched liquid collection tube was set to 0 mBar), and the sample was separated. After the separation, the second waste liquid collection tube and the enrichment liquid collection tube were collected separately, and then transferred to the orifice plate after centrifugation, and observed and counted under a fluorescence microscope. A total of 5 groups of experiments were carried out, 3 times in each group, and the average value was taken. The results are shown in the following table.

It can be seen that with the increase of the pressure parameter, the separation time is shortened, but the cell capture efficiency can still be maintained above 95%.

Cell Capture Efficiency Verification

According to the above separation method, the air pressure separation parameters are as follows: the pressure of the sample tube is 629 mBar, the pressure of the first waste liquid collection tube is 470 mBar, the pressure of the solution tube is 448 mBar, and the pressure of the second waste liquid collection tube and the enrichment liquid collection tube are both 0 mBar , add a large number of different types of tumor cell lines or cell lines into the sample tube (excluding red blood cells and white blood cells), add rinsing solution to the first waste liquid collection tube, and add buffer solution to the solution tube for sample loading and separation Capture, after the separation, collect the first waste liquid collection tube, the second waste liquid collection tube and the enrichment liquid collection tube respectively, centrifuge and transfer to the orifice plate, observe and count under the microscope. Each group of experiments were measured 3 times, and the average value was taken. The results are shown in the following table.

细胞类型cell type	第一废液收集管细胞数The number of cells in the first waste collection tube	第二废液收集管细胞数Number of cells in the second waste collection tube	富集液管细胞数Number of enriched ductal cells	捕获效率（富集液细胞数/总细胞数）Capture efficiency (number of cells in enrichment solution/total number of cells)
K562K562	33	88	472472	97.7%97.7%
A549A549	77	1515	635635	96.7%96.7%
SH-SY5YSH-SY5Y	66	2020	697697	96.4%96.4%
MCF7MCF7	33	1111	764764	98.2%98.2%
BGC-823BGC-823	44	1313	832832	98.0%98.0%
HepG2HepG2	22	1010	737737	98.4%98.4%

It can be seen that for different types of tumor cell lines, the capture efficiency is above 96%.

Morphological verification of circulating tumor cells (CTC), circulating tumor cell clusters (CTC Cluster) and circulating fusion cells (CFC) in peripheral blood

Take 3 mL of peripheral blood from tumor patients and dilute to 6 mL of PBS solution. According to the above separation method, the air pressure separation parameters are as follows: the pressure of the sample tube is 629 mBar, the pressure of the first waste liquid collection tube is 470 mBar, the pressure of the solution tube is 448 mBar, and the pressure of the second tube is 448 mBar. The air pressure of the waste liquid collection tube and the enrichment liquid collection tube are both 0 mBar. The first waste liquid collection tube is added with rinsing solution, and the buffer solution is added to the solution tube to carry out sample loading, separation and capture. After the separation, the enrichment liquid collection tube is taken. , centrifuged and transferred to a well plate, and stained with immunofluorescent antibody reagents, observed and counted under a microscope. Figure 4 is the cell morphology of circulating tumor cells (CTC), circulating tumor cell clusters (CTC Cluster) and fusion cells (CFC) observed under a fluorescence microscope, the red is the leukocyte CD45 staining, and the green is the tumor cell marker CK staining , the blue is DAPI staining of the nucleus, the definition of CTC is DAPI+/CD45-/CK+ cells, the circulating tumor cell mass is the cell mass formed by the aggregation of CTCs, the definition of fusion cell is DAPI+/CD45+/CK+, and tumor immune related cells.

Validation of CTCs, CTC Cell Clusters, and Fusion Cells (CFCs) Capture in Lung Cancer Peripheral Blood Samples

Take 5 mL of peripheral blood samples from 20 patients with lung cancer and dilute to 9 mL of PBS solution according to the above separation method and parameters: the pressure of the sample tube is 629 mBar, the pressure of the first waste liquid collection tube is 470 mBar, the pressure of the solution tube is 448 mBar, and the pressure of the second tube is 448 mBar. The pressure of the waste liquid collection tube and the enrichment liquid collection tube were both 0 mBar, and the sample was separated. After the separation, the enrichment liquid collection tube was collected, centrifuged and transferred to the orifice plate. After immunofluorescence antibody staining, it was observed and counted under a microscope, and the result was The detection data of circulating tumor cells (CTCs), circulating tumor cell clusters and fused cells (CFCs) in lung cancer samples are shown in the table below. Lung cancer CTCs are defined as DAPI+/CD45-/CK+ cells, circulating tumor cell clusters are cell clusters formed by the aggregation of CTCs, and fusion cells are defined as DAPI+/CD45+/CK+ cells that are related to tumor immunity.

样品sample	CTC数量Number of CTCs	CTC细胞团数量Number of CTC cell clusters	融合细胞（CFC）数量Confluent cell (CFC) number
11	11	00	1212
22	00	00	290290
33	33	00	55
44	33	00	11
55	00	00	22
66	00	00	1010
77	88	00	1212
88	33	00	66
99	44	00	305305
1010	00	00	66
1111	11	00	00
1212	00	00	33
1313	1414	00	55
1414	11	00	00
1515	11	00	33
1616	3333	2020	5252
1717	33	00	33
1818	99	00	66
1919	11	00	00
2020	55	00	99

It can be seen that in the peripheral blood samples of lung cancer, the number of fused cells (CFCs) > the number of CTCs > the number of CTC cell clusters.

Validation of capture of CTCs, CTC cell clusters and fused cells (CFCs) in peripheral blood samples from colorectal cancer

Take 5 mL of peripheral blood samples from 20 patients with colorectal cancer and dilute to 9 mL of PBS solution according to the above separation method and parameters: the pressure of the sample tube is 629 mBar, the pressure of the first waste liquid collection tube is 470 mBar, the pressure of the solution tube is 448 mBar, and the pressure of the first waste liquid collection tube is 470 mBar. The pressure of the second waste liquid collection tube and the enrichment liquid collection tube were both 0 mBar, and the sample loading and separation were carried out. After the separation, the enrichment liquid collection tube was collected, centrifuged and transferred to the orifice plate. After immunofluorescence antibody staining, the counts were observed under the microscope. The detection data of circulating tumor cells (CTC), circulating tumor cell clusters and fused cells (CFC) in intestinal cancer were obtained, as shown in the table below. Colorectal cancer CTCs are defined as DAPI+/CD45-/CK+ cells, circulating tumor cell clusters are cell clusters formed by the aggregation of CTCs, and fusion cells are defined as DAPI+/CD45+/CK+ cells that are related to tumor immunity.

样品sample	CTC数量Number of CTCs	CTC细胞团数量Number of CTC cell clusters	融合细胞（CFC）数量Confluent cell (CFC) number
11	00	00	22
22	66	00	33
33	22	00	22
44	55	00	99
55	00	00	1313
66	44	00	55
77	00	00	11
88	11	00	2727
99	11	00	33
1010	11	00	00
1111	00	00	55
1212	11	00	00
1313	33	00	11
1414	00	00	22
1515	00	00	11
1616	00	00	44
1717	22	00	22
1818	11	00	11
1919	33	00	00
2020	00	00	22

It can be seen that in the peripheral blood samples of colorectal cancer, the number of fused cells (CFCs) > the number of CTCs > the number of CTC cell clusters.

Validation of capture of CTCs, CTC cell clusters and fused cells (CFCs) in neuroblastoma peripheral blood samples

Take 3 mL of peripheral blood samples from 20 neuroblastoma patients, dilute to 6 mL of PBS solution, and follow the above separation method and parameters: the pressure of the sample tube is 629 mBar, the pressure of the first waste liquid collection tube is 470 mBar, and the pressure of the solution tube is 448 mBar , the pressure of the second waste liquid collection tube and the enrichment liquid collection tube are both 0 mBar, carry out sample loading and separation, collect the enrichment liquid collection tube after the separation, transfer to the well plate after centrifugation, and observe under the microscope after immunofluorescence antibody staining Counting to obtain the detection data of circulating tumor cells (CTC), circulating tumor cell clusters and fusion cells (CFC) of neuroblastoma samples, as shown in the table below. Neuroblastoma CTCs are defined as DAPI+/CD45-/GD2+ cells, circulating tumor cell clusters are cell clusters formed by the aggregation of CTCs, and fusion cells are defined as DAPI+/CD45+/GD2+ cells that are related to tumor immunity .

样品sample	CTC数量Number of CTCs	CTC细胞团数量Number of CTC cell clusters	融合细胞（CFC）数量Confluent cell (CFC) number
11	121121	00	146146
22	22	00	88
33	00	00	33
44	11	00	6161
55	00	00	22
66	00	00	2727
77	00	00	66
88	1414	00	1717
99	7272	24twenty four	00
1010	00	00	2727
1111	22	00	99
1212	33	00	88
1313	00	00	11
1414	193193	1010	105105
1515	1616	00	23twenty three
1616	00	00	22twenty two
1717	55	00	5252
1818	00	00	1010
1919	00	00	2727
2020	66	00	3838

It can be seen that in neuroblastoma peripheral blood samples, the number of fused cells (CFC) > the number of CTCs > the number of CTC cell clusters, and the number of fused cells (CFC) is usually higher than that of adult tumors, which may be related to Children's immune systems are different from adults.

The above embodiments are only used to illustrate the technical solutions of the present invention, not to limit the patent scope of the present invention. Any equivalent implementation or modification that does not depart from the present invention should be included within the scope of the technical solutions of the present invention.

Claims

A circulating tumor/fused cell capture device based on a microfluidic chip, characterized in that the capture device comprises a microfluidic chip used for cell separation and capture of a sample, and is sealed and connected to the microfluidic chip A control system for separating and capturing cells by providing a fluid driving force to a microfluidic chip; the control system includes an air source device and an air pressure connected to the air source device for controlling the output air pressure of the air source device A regulating device and a human-computer interaction device for setting and displaying the output air pressure; the human-computer interaction device is connected to the air pressure regulating device through a circuit control device.
The circulating tumor/fused cell capture device based on a microfluidic chip according to claim 1, wherein the capture device further comprises a device for loading samples or solutions used in conjunction with the microfluidic chip. container.
The device for capturing circulating tumor/fused cells based on a microfluidic chip according to claim 2, wherein the container comprises a sample tube for providing the sample to be separated to the microfluidic chip, a sample tube for The first waste liquid collection tube for collecting the main waste liquid after the primary separation of the microfluidic chip, the solution tube for providing the secondary separation solution and the hydrodynamic force to the sample after the primary separation and removal of the main waste liquid, for The second waste liquid collection tube for collecting the secondary waste liquid after the secondary separation of the microfluidic chip and the enrichment liquid collection tube for collecting the target cell enrichment liquid obtained after the secondary separation of the microfluidic chip, so The sample tube, the first waste liquid collection tube, the solution tube, the second waste liquid collection tube, and the enriched liquid collection tube are respectively sealed and connected to the corresponding inlet and outlet on the microfluidic chip through the conduit.
The device for capturing circulating tumor/fused cells based on a microfluidic chip according to claim 3, wherein the inlet and outlet include a sample inflow port corresponding to the sample tube and a first waste liquid collection The first waste liquid outflow port corresponding to the pipe, the solution inflow port corresponding to the solution pipe, the second waste liquid outflow port corresponding to the second waste liquid collection pipe, and the enrichment liquid outflow port corresponding to the enrichment liquid collection pipe.
The device for capturing circulating tumor/fused cells based on a microfluidic chip according to claim 4, wherein the operating table is provided with a tracheal interface device, one end of the tracheal interface device and the air pressure output of the air pressure regulating device The mouth is sealed and communicated, and the other end is sealed and communicated with the container.
The circulating tumor/fused cell capture device based on a microfluidic chip according to claim 1, wherein the microfluidic chip comprises a base layer and one or more structural layers disposed on the base layer, The structural layer is provided with a series of composite micro-pillar array structures composed of hydrodynamics and spatial deformation for enriching target cells.
The circulating tumor/fused cell capture device based on a microfluidic chip according to claim 6, wherein the microfluidic chip comprises a base layer, an upper structural layer and a lower structural layer, and the upper structural layer The layer is the guide layer, and the lower structure layer is the separation layer.
The device for capturing circulating tumor/fused cells based on a microfluidic chip according to claim 1, wherein the air pressure regulating device can be an electrical proportional valve.
The application of the microfluidic chip-based circulating tumor/fused cell capture device according to any of claims 1 to 8, wherein the capture device is used for:

(1) Isolation and/or capture of circulating tumor cells, circulating tumor cell clusters, and fused cells in peripheral blood samples; (2) Isolation and/or capture of pleural effusion, ascites, lymph, urine or bone marrow samples tumor cells, tumor cell clusters, and fusion cells;

(3) separation and/or capture of nucleated red blood cells in peripheral blood or umbilical cord blood samples;

(4) Isolation and/or capture of circulating endothelial cells in peripheral blood samples;

(5) Isolation and/or capture of leukocytes, T cells, B cells, lymphocytes, monocytes, granulocytes, natural Killer cells, dendritic cells, macrophages or hematopoietic stem cells;

(6) Separation and/or capture of red blood cells or platelets in peripheral blood, umbilical cord blood, pleural effusion, ascites, urine or bone marrow samples;

(7) Separation and/or capture of bacteria or viruses in peripheral blood, pleural effusion, ascites effusion, urine, saliva, plasma, serum, cerebrospinal fluid, semen, prostatic fluid or vaginal secretion samples;

(8) Isolation and/or capture of sperm in semen samples.
The method for capturing a circulating tumor/fused cell capture device based on a microfluidic chip according to any of claims 1 to 8, wherein the capturing method comprises the following steps:

(1) The blood sample to be separated is placed in the sample tube. The first waste liquid collection tube is filled with the rinsing solution for rinsing the chip and the catheter, and the solution tube is filled with the solution for rinsing and secondary separation of the sample. The liquid collection pipe and the enriched liquid pipe are empty pipes, and then a sealed channel is formed between the gas source device, each container and the chip;

(2) Set the air pressure of the first waste liquid collection tube, the sample tube, the solution tube, the second waste liquid collection tube and the enriched liquid collection tube at a certain time interval in turn, so that the rinsing in the first waste liquid collection tube The solution and the solution in the solution tube flow through the entire chip and pipeline, and play the role of rinsing and emptying the bubbles in the chip and pipeline;

(3) After the bubbles are discharged, set the air pressure of the sample tube, the first waste liquid collection tube, the solution tube, the second waste liquid collection tube and the enrichment liquid collection tube at the same time, so that the blood sample flows in from the chip inlet, buffering The liquid flows in from the solution inlet, the main waste blood produced after the primary separation flows out from the first waste liquid outlet, the secondary waste blood produced after the secondary separation flows out from the second waste liquid outlet, and the enriched liquid flows out of the enriched liquid outlet outflow;

(4) After the separation is completed, increase the pressure of the buffer pipeline, and adjust the pressure of the other pipelines to 0, so that the buffer solution fills the chip and all the thin tubes to prevent the pollution caused by the residual waste blood; Set to 0 to turn off.