Micro-fluidic chip integrating capture, culture and detection of circulating tumor cells and use method of micro-fluidic chip

Abstract

The invention relates to a microfluidic chip integrating capture, culture and detection of circulating tumor cells and a use method of the microfluidic chip. The microfluidic chip comprises an enrichment layer (100) and a culture layer (200), and the lower surface of the enrichment layer (100) is tightly attached to the upper surface of the culture layer (200); a sample inlet (11), a sample inlet pipeline (12), a sample outlet pipeline (13), a micro-column array (14) and a sample outlet (15) are arranged on the lower surface of the enrichment layer (100); the micro-column array (14) is positioned between the sample inlet pipeline (12) and the sample outlet pipeline (13) to separate the two pipelines; a microcavity array (21) is arranged on the upper surface of the culture layer (200). The CTCs can be enriched, cultured and detected on one chip, frequent manual transfer operation is avoided, and the activity and culture success rate of the CTCs are improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of microfluidic chips, and in particular relates to a microfluidic chip integrating the capture, culture and detection of circulating tumor cells and a method for using the same. Background Art

[0002] Circulating tumor cells (CTCs) are precursors to metastasis in various cancers and are considered the "seeds" of metastasis. They first detach from the margins of solid tumors, then enter the blood or lymphatic system, metastasizing from the primary site to distant organs via lymphatic channels or circulation. As tumor cells originating from primary or metastatic lesions, CTCs reflect the overall characteristics of invasive tumor cells in tumor tissues and possess many unique biological properties, offering tremendous research potential and broad clinical application value.

[0003] In recent years, organoids, as a new in vitro model, have revolutionized the study of disease basis and related transformations, and their application in the field of oncology has increased exponentially. Organoids can retain the histological and genetic characteristics of the primary tumor lesion and reproduce the heterogeneity within and between tumors. In addition, co-culturing organoids with primary stromal cells (such as fibroblasts, immune cells, etc.) can simulate the tumor microenvironment. Therefore, in addition to conventional chemotherapy drugs, they can also be used to screen immune drugs. The structure and function of organoids are highly similar to primary tumor tissues and are easy to operate. They are also widely used in basic research on tumor metastasis, drug resistance mechanisms, etc.

[0004] At present, most tumor organ chips are constructed based on existing tumor cell lines, which is not conducive to personalized research, so future organ chip platforms will be built on materials based on the patient himself. It is reported that the culture of tumor organoids from tumor tissue blocks is relatively easy to achieve, and 3D organoid culture has been preliminarily achieved in many organs such as the stomach, liver, breast, and colorectum. Compared with tumor tissue blocks, the acquisition of CTCs has the advantages of being non-invasive, simple to operate, repeatable sampling, and lower cost. However, due to the scarcity and heterogeneity of CTCs, the culture of CTC-derived organoids is extremely difficult. At present, only a few studies have reported the successful culture of CTCs organoids, but the success rate is very low, and most of them are from late-stage samples with very rich CTCs content (such as more than 100 CTCs per 8 ml of blood sample), while the success rate of organoid culture for early-stage tumors and samples with scarce CTCs content is extremely low. The main reasons for the low success rate of culture include two aspects: 1) the prolonged enrichment and manipulation of CTCs, which affects their survival; and 2) the complex and difficult to control in vitro culture environment and conditions for CTCs. Therefore, the development of high-success-rate CTC-derived tumor organoid culture technology is of great significance for studying tumor metastasis mechanisms, personalized drug sensitivity testing, and anti-tumor drug screening.

[0005] Currently, there are two reported CTC enrichment and culture techniques. One involves first enriching CTCs using membrane filtration (patents CN111197031 A; CN 114921414 A) or negative enrichment (patent CN 115125212 A), removing most white blood cells, and then transferring the enriched CTCs to microplates for culture. The other involves direct seeding of mononuclear cells, depleted of red blood cells, into microplates without enrichment (patents CN 109415702 A; CN 114874989 A). While both techniques can successfully culture CTCs, the success rate is relatively low. The former can easily lead to CTC loss or compromise CTC activity during transfer, while the latter, lacking an enrichment step, allows CTCs to be cultured with a large number of white blood cells, hindering the subsequent extraction of CTC organoids. Furthermore, both methods still require CTC transfer after successful culture for subsequent studies of tumor metastasis mechanisms or drug screening. During this transfer process, human manipulation can affect the activity of CTC organoids. In view of this, future CTCs culture technology should focus on how to preserve CTCs activity, reduce CTCs metastasis, and improve the success rate of CTCs culture. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a microfluidic chip integrating the capture, culture and detection of circulating tumor cells and its use method, so as to overcome the problem in the prior art that the activity of CTCs is reduced due to frequent manual transfer operations, thereby affecting the culture success rate.

[0007] The present invention provides a microfluidic chip that integrates the capture, culture and detection of circulating tumor cells. The microfluidic chip includes an enrichment layer and a culture layer, and the lower surface of the enrichment layer is tightly fitted with the upper surface of the culture layer; the lower surface of the enrichment layer is provided with an inlet, an inlet pipe, an outlet pipe, a microcolumn array and an outlet; the microcolumn array is located between the inlet pipe and the outlet pipe to separate the two pipes; the upper surface of the culture layer is provided with a microcavity array.

[0008] Preferably, the sample inlet end of the sample inlet pipe is open and the sample outlet end is closed; the sample inlet end of the sample outlet pipe is closed and the sample outlet end is open.

[0009] Preferably, the microcolumn array comprises microcolumns arranged at intervals; microfiltration channels are provided between the microcolumns; and the microfiltration channels are communicated with a sample inlet pipe and a sample outlet pipe.

[0010] Preferably, the microcavity array comprises microcavities arranged at intervals; the diameter of the microcavities is 100-800 μm and the depth is 100-800 μm.

[0011] Preferably, the microcavity array is neatly arranged on the center line of the sampling channel.

[0012] Preferably, the material of the microfluidic chip is any one of PDMS, PMMA, PC, and PP.

[0013] The present invention provides a method for using a microfluidic chip that integrates the capture, cultivation, and detection of circulating tumor cells, comprising the following steps:

[0014] S1. The collected blood sample is subjected to red blood cell lysis, and the cell pellet after centrifugation is resuspended in culture medium to obtain a cell suspension. S2. The cell suspension is introduced into the microfluidic chip from the sampling port via an injection pump. Smaller white blood cells flow out of the sample outlet channel through the microfiltration channel, while larger white blood cells (monocytes, macrophages, etc.) and circulating tumor cells (CTCs) are intercepted in the sampling channel by the microcolumn array and enter the microcavity array below the center of the sampling channel.

[0015] S3. Place the microfluidic chip in a culture dish and add culture medium until the chip is submerged. The dish is incubated at 37°C, 5% CO2, and 0-4% O2. Fresh culture medium is added to the chip every 8-24 hours to complete the culture of CTCs in the microcavity.

[0016] S4. Perform subsequent analysis and detection of CTCs in the microcavity according to application requirements.

[0017] Furthermore, the application requirements include but are not limited to:

[0018] (1) By applying different injection pressures and liquid perfusion rates, different blood flow shear forces in the blood vessels were simulated to study the effects of shear force on CTCs activity, proliferation ability, cell morphology, cell molecular phenotype, etc.

[0019] (2) Compare the activity, proliferation, and spheroidization ability of CTCs in the blood of cancer patients in different groups (before treatment vs. after treatment, different cancer types, different stages, non-metastatic vs. metastatic, etc.) to explore the mechanism of tumor metastasis.

[0020] (3) Different stromal cells (endothelial cells, fibroblasts, etc.) are added in reverse through the sample outlet. The stromal cells will be trapped in the sample outlet and co-cultured with CTCs and residual leukocytes to study the effects of different stromal cells and extracellular matrix on the proliferation, migration and invasion of CTCs; immune microspheres are introduced from the sample inlet to capture cell-secreted factors and exosomes in real time to study the mechanism of interaction between stromal cells and CTCs.

[0021] (4) By adding different chemotherapy drugs, the sensitivity of CTCs to drugs is detected for clinical screening of therapeutic drugs.

[0022] Beneficial effects

[0023] (1) The present invention can enrich, culture, and detect CTCs on a single chip, avoiding frequent manual transfer operations and improving the activity and culture success rate of CTCs.

[0024] (2) The chip provided by the present invention has multiple application scenarios, including but not limited to CTCs counting, CTCs typing, CTCs metastasis mechanism research, chemotherapy drug screening, etc.

[0025] (3) The chip provided by the present invention is based on microfluidic technology and has the advantages of small size, easy to carry, simple operation, low consumption of detection reagents, small sample volume requirement, and low detection cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Schematic diagram of the microfluidic chip of the present invention.

[0027] Figure 2 This is a partially enlarged 3D image of the sampling channel in the microfluidic chip of the present invention.

[0028] Figure 3 Schematic diagram of the principle of capturing CTCs using the microfluidic chip of the present invention.

[0029] Figure 4 AB are the results of H1975 cells culturing into spheres in the microcavity of the present invention.

[0030] Figure 5 This is a schematic diagram of the present invention wherein stromal cells are introduced from the sample outlet for co-culturing with CTCs.

[0031] Figure 6 Schematic diagram of the present invention using immune microspheres to capture secretions such as cell-secreted factors and exosomes. DETAILED DESCRIPTION

[0032] Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiments are only used to illustrate the present invention and are not used in limiting the scope of the present invention.In addition, should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall equally within the scope limited by the appended claims of the application.

[0033] Example 1

[0034] like Figure 1 and Figure 2FIG. 1 shows a microfluidic chip according to a preferred embodiment of the present invention. The microfluidic chip includes an enrichment layer 100 and a culture layer 200. The lower surface of the enrichment layer 100 is in close contact with the upper surface of the culture layer 200. The lower surface of the enrichment layer 100 is provided with an inlet 11, an inlet pipe 12, an outlet pipe 13, a micropillar array 14, and an outlet 15. The micropillar array 14 is located between the inlet pipe 12 and the outlet pipe 13, separating the two pipes. The upper surface of the culture layer 200 is provided with a microcavity array 21. The inlet end of the inlet pipe 12 is open, and the outlet end is closed. The inlet end of the outlet pipe 13 is closed, and the outlet end is open.

[0035] The micro-pillar array 14 includes micro-pillars 141 arranged at intervals. A micro-filtration channel 142 is provided between the micro-pillars. The micro-filtration channel 142 is communicated with the sample inlet pipe 12 and the sample outlet pipe 13 .

[0036] The microcavity array 21 includes microcavities arranged at intervals.

[0037] According to this preferred embodiment, the width of the sample inlet pipe 12 is 800 μm; the width of the sample outlet pipe 13 is 600 μm; the width of the microfiltration pipe 142 is 5-10 μm; the diameter of the microcavity is 100-800 μm, and the depth is 100-800 μm.

[0038] Example 2

[0039] According to Example 1, a microfluidic chip integrating CTCs capture, culture and detection is provided, and its processing and preparation method is as follows:

[0040] 2.1 Use AutoCAD software to draw the structures of the enrichment layer and culture layer respectively, and process and make the mask plates respectively.

[0041] 2.2 Using a four-inch single crystal silicon wafer as the substrate, the enrichment layer has a small microfiltration pipeline structure and requires high precision. Therefore, the silicon mold of the enrichment layer can be obtained by spin-coating positive resist on the silicon wafer, photolithography, development, deep reactive ion etching, and de-resin cleaning. The microcavity size of the culture layer is larger and does not require high precision. Therefore, the silicon mold of the culture layer can be obtained by spin-coating negative resist on the silicon wafer, photolithography, and development.

[0042] 2.3 Place the silicon mold and the glass slide with fluorosilane in a vacuum drying oven and apply negative pressure overnight. Remove the silicon wafer the next day. Fluorosilane makes the silicon wafer surface more hydrophobic, facilitating subsequent PDMS demolding.

[0043] 2.4 Weigh the PDMS prepolymer and curing agent (mass ratio 10:1) into a plastic cup, stir well, and place in a vacuum drying oven, apply negative pressure, and let it sit for 30-60 minutes. Pour the PDMS mixture onto a silicon mold and heat in a 60°C oven for 3-4 hours. Peel the PDMS chip from the silicon mold, cut it, and punch holes.

[0044] 2.5 Place the PDMS chip structure of the enrichment layer and the culture layer face up in a plasma cleaner and clean for 1 minute. After taking them out, align and bond them under a microscope so that the microcavity array of the culture layer is on the center line of the enrichment layer sampling channel. Place the bonded chip in a 60℃ oven and heat for 6-8 hours. The preparation of the microfluidic chip is completed.

[0045] Example 3

[0046] A method for enriching, culturing and detecting CTCs comprises the following steps:

[0047] 3.1 The chip prepared in Example 2 was soaked in anhydrous ethanol for 1 hour. After air-drying, 200 μL of 0.5% Pluronic F127 solution was introduced into the chip using a positive / negative pressure injection pump. The chip was then placed in a 37°C incubator for 0.5-1 hour. PBS buffer was then introduced into the chip to wash the internal channels and microcavities. The chip was then placed in a biosafety cabinet and exposed to UV light for 30 minutes. The introduction of F127 renders the microcavities hydrophobic, facilitating cell spheroid formation.

[0048] 3.2 The red blood cells in the collected blood samples from cancer patients were lysed using red blood cell lysis buffer (Beijing Solebow Technology Co., Ltd.). The cell pellet after centrifugation was resuspended in 200 μL of culture medium (1640 culture medium + 10% fetal bovine serum). The resulting cell suspension was introduced into the microfluidic chip through the sampling port 11 via a positive / negative pressure sampling pump. Smaller white blood cells flowed out of the sampling channel 13 through the microfiltration channel 142, while larger white blood cells and circulating tumor cells (CTCs) were intercepted by the micropillar array 14 in the sampling channel 12 and entered the microcavity array 21 below the center of the sampling channel 12 ( Figure 3 ).

[0049] 3.3 Place the chip in a 3-inch culture dish and add culture medium to the dish until the chip is covered. Place the dish in a 37°C, 5% CO2, and 0-4% O2 environment and add fresh culture medium to the chip every 8-24 hours.

[0050] 3.4 After 2-4 weeks of culture, 1% polymethanol solution was passed into the chip and incubated at room temperature for 10 minutes to fix the cells. The polymethanol solution was removed and cell permeabilization and blocking solution was added. The cells were incubated at room temperature for 15 minutes for permeabilization and blocking. Antibody mixture (CD45 and CK antibodies, DAPI solution) was added and incubated at 37°C in the dark for 1 hour. The antibodies were removed and the cells were rinsed 3 times with washing solution. The cultured CTCs ( Figure 4 ).

[0051] Example 4

[0052] application:

[0053] 4.1 By applying different injection pressures and liquid perfusion rates, different blood flow shear forces in the blood vessels were simulated to study the effects of shear force on CTCs activity, proliferation ability, cell shape, cell molecular phenotype, etc.

[0054] 4.2 Compare the activity, proliferation, and spheroidization of CTCs in the blood of cancer patients in different groups (before treatment vs. after treatment, different cancer types, early, mid-late stage, non-metastatic vs. metastatic, etc.) to explore the mechanism of tumor metastasis.

[0055] 4.3 Add different stromal cells (endothelial cells, fibroblasts, etc.) through the sample outlet. The stromal cells will be trapped in the sample outlet and co-cultured with CTCs and residual white blood cells ( Figure 5 ) to study the effects of different stromal cells and extracellular matrices on CTCs proliferation, migration and invasion; introduce immune microspheres from the injection port to capture cell secretion factors and exosomes in real time ( Figure 6 ), to study the mechanism of interaction between stromal cells and CTCs.

[0056] 4.4 By adding different chemotherapy drugs, the sensitivity of CTCs to drugs is detected for clinical screening of therapeutic drugs.

Claims

1. A microfluidic chip integrating the capture, culture, and detection of circulating tumor cells, characterized by: The microfluidic chip comprises an enrichment layer (100) and a culture layer (200), wherein the lower surface of the enrichment layer (100) and the upper surface of the culture layer (200) are in close contact with each other; the lower surface of the enrichment layer (100) is provided with an inlet (11), an inlet pipe (12), an outlet pipe (13), a microcolumn array (14), and an outlet (15); the microcolumn array (14) is located between the inlet pipe (12) and the outlet pipe (13), separating the two pipes; and the upper surface of the culture layer (200) is provided with a microcavity array (21).

2. The microfluidic chip according to claim 1, wherein: The sample inlet end of the sample inlet pipe (12) is open, and the sample outlet end is closed; the sample outlet end of the sample outlet pipe (13) is closed, and the sample outlet end is open.

3. The microfluidic chip according to claim 1, wherein: The microcolumn array (14) comprises microcolumns (141) arranged at intervals; a microfiltration channel (142) is provided between the microcolumns; and the microfiltration channel (142) is communicated with a sample inlet pipe (12) and a sample outlet pipe (13).

4. The microfluidic chip according to claim 1, wherein: The microcavity array (21) comprises microcavities arranged at intervals; the diameter of the microcavities is 100-800 μm and the depth is 100-800 μm.

5. The microfluidic chip according to claim 1, wherein: The microcavity array (21) is neatly arranged on the center line of the sampling pipe (12).

6. The microfluidic chip according to claim 1, characterized in that: The material of the microfluidic chip is any one of PDMS, PMMA, PC and PP.

7. A method for using the microfluidic chip for capturing, culturing, and detecting circulating tumor cells according to any one of claims 1 to 6, comprising the following steps: S1. Lyse the red blood cells of the collected blood sample, and resuspend the cell pellet after centrifugation in culture medium to obtain a cell suspension; S2. The cell suspension is introduced into the microfluidic chip from the injection port (11) through the injection pump. The smaller white blood cells flow out from the sample outlet pipe (13) through the microfiltration channel (142). The larger white blood cells and circulating tumor cells (CTCs) are intercepted by the microcolumn array (14) in the injection pipe (12) and enter the microcavity array (21) below the center of the injection pipe (12). S3. Place the microfluidic chip in a culture dish and add culture medium until the chip is submerged. The dish is incubated at 37°C, 5% CO2, and 0-4% O2. Fresh culture medium is added to the chip every 8-24 hours to complete the culture of CTCs in the microcavity. S4. Perform subsequent analysis and detection of CTCs in the microcavity according to application requirements.

Images