Micro-fluidic chip for simulating tumor metastasis cascade process and preparation method of micro-fluidic chip

Abstract

The invention belongs to the field of biomedical engineering, and relates to a microfluidic chip for simulating a tumor metastasis cascade process, which sequentially comprises a substrate (1), a middle-layer porous film (2) and a cover plate (3) from bottom to top, a U-shaped channel (4) is formed in one surface, close to the middle-layer porous film (2), of the substrate (1); a left side channel (5) and a right side channel (6) are formed in one surface, adjacent to the middle-layer porous film (2), of the cover plate (3); the substrate (1), the middle-layer porous film (2) and the cover plate (3) are tightly bonded, the left side channel (5) is overlapped with a left side channel of the U-shaped channel (4), and the right side channel (6) is overlapped with a right side channel of the U-shaped channel (4). The micro-fluidic chip disclosed by the invention can be used for simulating a tumor primary focus and a metastasis microenvironment, and simulating a whole tumor metastasis cascade process including a plurality of stages of tumor cell infiltration, circulation, extravasation and colonization in vitro.

Description

Technical Field

[0001] This invention relates to the field of biomedical engineering, and in particular to a microfluidic chip that simulates the cascade process of tumor metastasis and its preparation method. Background Technology

[0002] Tumor metastasis is a major cause of poor prognosis and disease recurrence and death in cancer patients. The tumor metastatic cascade describes the complex, multi-step process by which malignant tumor cells spread from the primary site to distant organs and form secondary tumors (metastases). This process involves the shedding, infiltration, circulation, extravasation, and colonization of cancer cells. It is not random but rather a journey in which cancer cells overcome numerous physiological barriers, actively adapt to and modify the microenvironment, and invade distant organs. Hematogenous metastasis of tumor cells is one of the most common pathways of tumor metastasis, involving multiple complex steps such as tumor cell shedding from the primary lesion, infiltration into the bloodstream, extravasation from blood vessels, and colonization in other organs to form metastases. These steps are interconnected and collectively drive disease progression. Comprehensive simulation and research of the tumor metastasis cascade process is crucial for a deeper understanding of cancer metastasis biology, the discovery of new diagnostic biomarkers and therapeutic targets, and the development of effective anti-metastatic treatment strategies, which is of great significance for advancing the development of cancer therapy and the pharmaceutical industry.

[0003] Traditional methods for simulating tumor metastasis primarily rely on in vivo animal models and Transwell assays. Transwell assays are powerful and practical in vitro tools for studying cell migration and invasion, particularly suitable for high-throughput screening. However, their significant limitation lies in the vast difference between the highly simplified in vitro environment and the actual physiological / pathological state in vivo. They cannot accurately simulate the complex tumor microenvironment and metastasis process in vivo, and are unsuitable for real-time dynamic monitoring, thus limiting their application in biomedical research and in-depth exploration of tumor metastasis. Animal models, such as mouse models, involve ethical controversies, exhibit species differences from humans, and have long experimental cycles and high costs.

[0004] Microfluidics is a science, technology, and engineering discipline involving the precise manipulation and processing of extremely small volumes (picoliters to microliters) of fluid within a network of channels at the micrometer scale (typically tens to hundreds of micrometers). It is also known as "lab-on-a-chip" or "micro total analysis system." In recent years, microfluidics has gained significant traction due to its advantages, including precise fluid manipulation, simulation of in vivo mechanical forces and biochemical environments, ease of integration, high throughput, and real-time monitoring. These advantages enable the reproduction of key physiological characteristics and functions of organs or tissues on microchips, providing a highly biomimetic experimental platform for simulating and studying tumor metastasis. Currently, microfluidics is frequently used to simulate the tumor microenvironment and construct tumor metastasis models, making progress in exploring the mechanisms of tumor metastasis. However, current tumor metastasis models still have limitations: they can only simulate the local tumor microenvironment and single stages of the tumor metastasis process, lacking a systematic simulation of the entire metastasis process.

[0005] For example, Ingber's team at Harvard University designed a bilayer microfluidic chip to construct an in situ lung cancer chip model, simulating the invasion process of lung cancer cells. Chen et al. developed an organoid chip platform integrating hexagonal Transwell chambers (with porous membranes on the sidewalls of the chambers), cultured melanoma organoids in the middle of the chambers, and monitored the migration process of tumor cells through the matrix and porous membranes, simulating the horizontal migration process of tumor cells into surrounding tissues.

[0006] Patent document CN104830683A discloses a biomimetic microfluidic chip for simulating the microenvironment of tumor cells and their metastasis in vivo. The chip consists of a three-layer PDMS substrate and two-layer porous PDMS membranes. A blood-air barrier is constructed by two-dimensionally culturing bronchial epithelial cells, vascular endothelial cells, macrophages, and fibroblasts on the upper and lower surfaces of the membranes, respectively. Two vacuum channels are connected to both sides perpendicular to the membrane to simulate the contraction and expansion of alveoli, thus constructing a lung-on-a-chip physiological model. Based on this, lung cancer cells are seeded into the bronchial epithelial cell region, and culture chambers for glial cells, osteoblasts, and hepatocytes are constructed to simulate the metastasis process of lung cancer cells to specific target organs. This model is designed based on common origins and metastatic sites of lung cancer, thus omitting other lung cancer origins and other lung cancer metastasis pathways. The model is complex to construct and difficult to manufacture, and it does not consider the influence of fluid environments such as blood and lymph on tumor metastasis, only simulating the invasion process of tumor cells to specific target organs. The article "Oxygen Concentration Gradient Microfluidic Chip for Tumor Metastasis Research" published by Sun Dexian et al. of Dalian University of Technology designed and fabricated a tumor metastasis microfluidic chip with an integrated oxygen concentration gradient generator. From top to bottom, it includes: a PC membrane to prevent oxygen from entering or leaving, a cavity for oxygen to enter, a PDMS diffusion layer, a culture medium layer, a cell layer, and a bottom oxygen-impermeable glass layer. However, it only simulates the changes in the oxygen concentration microenvironment during tumor metastasis and lacks a systematic simulation of the entire metastasis process.

[0007] In summary, there is still an urgent need to develop a microfluidic device that can realistically simulate the entire process of tumor metastasis cascade in vivo, in order to further explore the mechanisms of cancer metastasis and anti-metastasis methods. Summary of the Invention

[0008] The purpose of this invention is to provide a microfluidic chip that simulates the tumor metastasis cascade process and its preparation method. The microfluidic chip of this invention can simulate the primary tumor lesion and metastatic microenvironment, and simulate the entire tumor metastasis cascade process in vitro, including multiple stages such as tumor cell infiltration, circulation, extravasation and colonization. It does not limit the origin of tumor cells and the metastatic pathway, and more realistically approximates the in vivo metastatic microenvironment to simulate the tumor metastasis cascade process.

[0009] To address the aforementioned technical problems, the microfluidic chip simulating the tumor metastasis cascade process and its fabrication method provided by this invention are implemented as follows:

[0010] A microfluidic chip simulating the cascade process of tumor metastasis comprises, from bottom to top: a substrate 1, a porous membrane 2, and a cover plate 3; a U-shaped channel 4 is provided on the side of the substrate 1 that is attached to the porous membrane 2, and a left channel 5 and a right channel 6 are provided on the side of the cover plate 3 that is attached to the porous membrane 2; the substrate 1, the porous membrane 2, and the cover plate 3 are attached together, the left channel 5 overlaps with the left channel of the U-shaped channel 4, and the right channel 6 overlaps with the right channel of the U-shaped channel 4; the left channel 5 is used for culturing tumor cells, the right channel 6 provides space for tumor cell extravasation and colonization, and the U-shaped channel 4 simulates the structure and function of blood vessels and the fluid environment, providing environmental conditions for tumor cell infiltration, circulation, and extravasation; tumor cells in the left channel 5 can migrate through the channel and the porous membrane in the U-shaped channel 4 and the right channel 6.

[0011] This invention features a U-shaped channel 4, a left-side channel 5, and a right-side channel 6. The system simulates the microenvironment of the primary tumor tissue and the metastatic process. Tumor cells in the left-side channel 5 can migrate within the U-shaped channel 4 and the right-side channel 6, comprehensively simulating the entire metastatic cascade process of tumor cell invasion, infiltration, circulation, extravasation, and colonization. Compared to existing technologies, the microfluidic chip in this invention is suitable for simulating the entire metastatic cascade process of various cancers, more realistically approximating the actual blood metastasis process of cancer in vivo. The chip has a simple structure, is easy to manufacture, low in cost, easy to operate, and less prone to problems during use.

[0012] Optionally, the intermediate porous film 2 is made of porous polycarbonate or polyethylene terephthalate, and micropores with a pore size between 5 and 15 μm are uniformly distributed on the intermediate porous film 2.

[0013] Optionally, the substrate 1 and the cover plate 3 are made of light-transmitting polydimethylsiloxane.

[0014] The substrate and cover plate of this invention, as well as the U-shaped channel, left channel and right channel thereon, are made of light-transmitting polydimethylsiloxane. When labeling tumor cells in the left channel and vascular endothelial cells in the U-shaped channel, it is convenient to observe the movement direction of different markers and to realize the observation and counting of metastatic tumor cells.

[0015] Optionally, the height of the U-shaped channel 4 is 80–300 μm and the width is 0.5–3 mm; the height of the left channel 5 is 200–500 μm and the width is 0.5–3 mm; the dimensions of the right channel 6 are the same as those of the left channel 5; the length of the overlap between the left channel 5, the right channel 6 and the U-shaped channel 4 is 2–10 mm.

[0016] This invention restricts the height and width of the channels to simulate the microenvironment of capillaries and lymphatic vessels within tumor tissue, better mimicking cell migration behavior within blood vessels. The height and width of the U-shaped channels support cell cluster manipulation and low-shear perfusion, avoiding the clogging problems of traditional microchannels. The limited length of the overlapping area ensures that the dynamic process of migration across barriers is observable. The left and right channels are slightly taller to simulate the microenvironment of primary or metastatic tumor lesions.

[0017] Optionally, the U-shaped channel 4 has an inlet 41 and an outlet 42 at both ends, the left channel 5 has an inlet 51 and an outlet 52 at both ends, and the right channel 6 has an inlet 61 and an outlet 62 at both ends; the inlet 41 and outlet 42, the inlet 51 and outlet 52, and the inlet 61 and outlet 62 are all provided on the cover plate 3.

[0018] The inlet and outlet of the channel are both located on the cover plate 3, which facilitates the introduction of solution or cells, or provides shear stress.

[0019] This invention also provides a method for fabricating a microfluidic chip that simulates the cascade process of tumor metastasis, comprising the following steps:

[0020] Molds for fabricating microfluidic chip substrates and cover plates based on standard photolithography: The substrate and cover plate molds are fabricated on a clean silicon wafer using SU-8 photoresist. The substrate mold contains a model of a U-shaped channel 4, and the cover plate mold contains a model of a left channel 5 and a right channel 6.

[0021] A polydimethylsiloxane solution was prepared and poured into the base mold and the cover plate mold, respectively, and then heated to cure.

[0022] Cut the base and cover plate with channels from the mold surface, and use a manual punch to make liquid inlet 41 and liquid outlet 42, liquid inlet 51 and liquid outlet 52, and liquid inlet 61 and liquid outlet 62 at the corresponding positions on the upper cover.

[0023] By using plasma treatment or polydimethylsiloxane solution as an adhesive, the side of the substrate with U-shaped channels is first sealed to the middle porous film, and then the other side of the middle porous film is bonded to the cover plate to obtain the microfluidic chip simulating the tumor metastasis cascade process.

[0024] Optionally, the preparation of the polydimethylsiloxane solution involves adding a crosslinking agent to the polydimethylsiloxane monomer, wherein the mass ratio of the polydimethylsiloxane monomer to the crosslinking agent is 5 to 15:1.

[0025] Optionally, the temperature for heat curing is 50–80°C.

[0026] A method for simulating and monitoring the tumor metastasis cascade process using a microfluidic chip includes the following steps:

[0027] After cleaning and sterilizing the microfluidic chip, a matrix protein solution is introduced into the U-shaped channel 4. The microfluidic chip is then placed in a 37°C constant temperature incubator and incubated for 10 min to 2 h. The microfluidic chip is then flipped over and incubated again to uniformly coat the surface of the U-shaped channel 4 with a thin layer of matrix protein.

[0028] A suspension of vascular endothelial cells labeled with a live cell tracer is introduced into the U-shaped channel 4 coated with the matrix protein. The microfluidic chip is flipped and placed in an incubator to stand for 2-12 hours to allow the cells to adhere to the walls of the U-shaped channel 4. The U-shaped channel 4 is then filled with vascular endothelial cell culture medium and cultured for another 6-24 hours to allow the vascular endothelial cells to stabilize and form tight junctions.

[0029] A matrix gel solution of resuspended tumor cells labeled with live cell tracer was introduced into the left channel 5. The microfluidic chip was placed in a 37°C incubator and incubated for 10-30 minutes to solidify the matrix gel of the resuspended tumor cells. Tumor cell-specific culture medium was added into the left channel 5.

[0030] A special culture medium containing biomolecules that can affect the migration of tumor cells is added into the right channel 6; a peristaltic pump is used to apply fluid shear stress stimulation in the U-shaped channel 4.

[0031] The microfluidic chip treated as described above was placed in an incubator for culture. The tumor cell culture medium in the left channel 5 was replaced every 12 to 48 hours. The migration and location of tumor cells in the microfluidic chip channel were observed and recorded in real time under a fluorescence microscope every 2 to 12 hours. The number of tumor cells that migrated into the U-shaped channel 4 and invaded the right channel 6 were counted to simulate and monitor the tumor metastasis cascade process.

[0032] In this invention, vascular endothelial cells and tumor cells are labeled with different colored live cell tracers, enabling clear differentiation of tumor cell migration. The matrix gel solution resuspending tumor cells includes single tumor cells or tumor cell spheres.

[0033] Optionally, the special culture medium contains biomolecules that can affect tumor cell migration, including high concentrations of one or more of serum, cytokines, growth factors, and chemokines.

[0034] Different biomolecules are selected based on the type of tumor cell. The U-shaped channel is not limited to vascular endothelial cells; cells or matrix proteins that conform to the tumor metastasis microenvironment can be selected as needed. The matrix proteins provided in this invention include one or more of the following: collagen, fibronectin, laminin, gelatin, matrix gum, and polylysine. Tumor cells can also be selected from various cancer cells such as breast cancer, lung cancer, liver cancer, colorectal cancer, and pancreatic cancer.

[0035] The present invention has the following beneficial effects:

[0036] The microfluidic chip of this invention integrates the simulation of the entire process of tumor metastasis, including the primary tumor lesion and multiple stages such as tumor cell infiltration, circulation, extravasation and colonization. It realistically approximates the in vivo metastasis microenvironment, can be adapted to different types of cancer, and provides a promising platform for exploring the mechanism of tumor metastasis and screening anti-metastasis drugs.

[0037] Furthermore, the microfluidic chip of this invention consists of only three layers: upper, middle, and lower. It has three channels only in the upper and lower layers, making it simple in structure, easy to manufacture, and less prone to failure during use. Attached Figure Description

[0038] Figure 1 This is a structural diagram of the microfluidic chip used in this invention to simulate the tumor metastasis cascade process; Figure 1 (A) is a schematic diagram of the overall structure of the microfluidic chip; Figure 1 (B) is a schematic diagram of the layered structure of a microfluidic chip;

[0039] Figure 2 This is a flowchart illustrating the fabrication process of the chip substrate and chip cover of the microfluidic chip in this invention.

[0040] Figure 3This is a schematic diagram of the mask used in the fabrication of the microfluidic chip in this invention; Figure 3 (A) is a schematic diagram of the mask of the underlying substrate of the chip; Figure 3 (B) is a schematic diagram of the mask on the top cover of the chip;

[0041] Figure 4 These are physical images and U-shaped channel characterization diagrams of the chip substrate prepared according to Embodiment 1 of the present invention. Figure 4 (A) is a physical image of the substrate beneath the chip; Figure 4 (B) is a magnified microscope image of a U-shaped channel in the underlying substrate of the chip; Figure 4 (C) is a cross-sectional view of the U-shaped channel in the underlying substrate of the chip; where Figure 4 (A) Add red pigment water into the U-shaped channel. Figure 4 (B) and Figure 4 (C) shows the actual width and height of the U-shaped channel;

[0042] Figure 5 This is a physical image of the chip cover sheet prepared according to Embodiment 1 of the present invention, and characterization diagrams of two channels therein; Figure 5 (A) is a physical image of the chip's top cover plate; Figure 5 (B) is a magnified microscope image of the left channel in the upper cover of the chip; Figure 5 (C) is a cross-sectional view of the left channel; where Figure 5 Add red dye solution to the left and right channels in (A). Figure 5 (B) and Figure 5 (C) shows the actual width and height of the left and right passageways, respectively;

[0043] Figure 6 These are physical images and cross-sectional views of the microfluidic chip prepared according to Embodiment 1 of the present invention; Figure 6 (A) is a physical image of the microfluidic chip and an enlarged view of the corresponding area; Figure 6 (B) is a cross-sectional view of the microfluidic chip;

[0044] Figure 7 This is a schematic diagram of the microfluidic chip prepared according to Embodiment 1 of the present invention, through which different colored pigment aqueous solutions are introduced;

[0045] Figure 8 This is a flowchart illustrating the simulation and monitoring of the tumor metastasis cascade process based on the microfluidic chip in this invention;

[0046] Wherein: 1—substrate, 2—middle porous membrane, 3—cover sheet, 4—U-shaped channel, 5—left channel, 6—right channel, 41—U-shaped channel inlet, 42—U-shaped channel outlet, 51—left channel inlet, 52—left channel outlet, 61—right channel inlet, 62—right channel outlet. Detailed Implementation

[0047] To make the objectives, technical solutions, and advantages of this invention clearer, the following embodiments provide a more detailed description of the invention. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of the invention.

[0048] like Figure 1 , Figure 2 and Figure 3 As shown, a microfluidic chip simulating the cascade process of tumor metastasis includes, from bottom to top: a substrate 1, a porous membrane 2, and a cover plate 3; a U-shaped channel 4 is provided on the side of the substrate 1 near the porous membrane 2, and a left channel 5 and a right channel 6 are provided on the side of the cover plate 3 near the porous membrane 2; the substrate 1, the porous membrane 2, and the cover plate 3 are attached together, the left channel 5 overlaps with the left channel of the U-shaped channel 4, and the right channel 6 overlaps with the right channel of the U-shaped channel 4; the left channel 5 is used for tumor cell culture, the right channel 6 provides space for tumor cell extravasation and colonization, and the U-shaped channel 4 simulates vascular structure and fluid environment, providing environmental conditions for tumor cell infiltration, circulation, and extravasation; tumor cells in the left channel 5 can migrate through the channel and the porous membrane in the U-shaped channel 4 and the right channel 6.

[0049] This invention features a U-shaped channel 4, a left-side channel 5, and a right-side channel 6. Tumor cells in the left-side channel 5 can migrate through the U-shaped channel 4 and the right-side channel 6, simulating the primary and metastatic microenvironments of the tumor. This comprehensively simulates the entire metastatic cascade process of tumor cell invasion, infiltration, circulation, extravasation, and colonization. Compared to existing technologies, the microfluidic chip in this invention is suitable for simulating the entire metastatic cascade process of various cancers, more realistically approximating the actual blood metastasis process of cancer in vivo. The chip has a simple structure, is easy to manufacture, low in cost, easy to operate, and less prone to problems during use.

[0050] The middle porous film 2 is made of polycarbonate or polyethylene terephthalate, and micropores with a pore size between 5 and 15 μm are uniformly distributed on the middle porous film 2.

[0051] Optionally, the substrate 1 and the cover plate 3 are made of light-transmitting polydimethylsiloxane, the U-shaped channel 4 is made of the same material as the substrate 1, and the left channel 5 and the right channel 6 are made of the same material as the cover plate 3.

[0052] The substrate and cover plate of this invention, as well as the U-shaped channel, left channel and right channel thereon, are made of light-transmitting polydimethylsiloxane. When labeling tumor cells in the left channel and vascular endothelial cells in the U-shaped channel, it is convenient to observe the movement direction of different markers and to count metastatic tumor cells.

[0053] The U-shaped channel 4 includes a straight inlet channel with a width of 0.5 to 3 mm and a height of 80 to 300 μm, a 180° semi-circular curved channel with a radius of curvature (R) of 0.25 to 1.5 mm, and a straight outlet channel parallel to the inlet channel; the three channels are interconnected and have the same width and height.

[0054] Left channel 5 and right channel 6 are parallel to each other, with identical dimensions (width × height: 0.5–3 mm × 200–500 μm), and each has an independent inlet / outlet for independent perfusion. Spatially, left channel 5 and right channel 6 overlap with the straight inlet and outlet channels of the lower U-shaped channel, with an overlap length of 2–10 mm. The two channels in the overlapping area are separated by a porous membrane. Its core function is that the porous membrane, with its micron-scale pore structure, allows for material diffusion and exchange between the channels on both sides of the membrane, generating a stable concentration gradient within the three channels of the chip; it also facilitates intercellular communication across the membrane, providing conditions for observing and studying tumor cell migration.

[0055] Channel height refers to the dimension perpendicular to the substrate plane, while width is the lateral dimension parallel to the chip substrate plane.

[0056] This invention restricts the height and width of the channels to simulate the microenvironment of capillaries and lymphatic vessels within tumor tissue, better mimicking cell migration behavior within blood vessels. The height and width of the U-shaped channels support cell cluster manipulation and low-shear perfusion, avoiding the clogging problems of traditional microchannels. The limited length of the overlapping area ensures that the dynamic process of migration across barriers is observable. The left and right channels are slightly taller to simulate the microenvironment of primary or metastatic tumor lesions.

[0057] The U-shaped channel 4 of the present invention has an inlet 41 and an outlet 42 at both ends, the left channel 5 has an inlet 51 and an outlet 52 at both ends, and the right channel 6 has an inlet 61 and an outlet 62 at both ends; the inlet 41 and outlet 42, the inlet 51 and outlet 52, and the inlet 61 and outlet 62 are all provided on the cover plate 3.

[0058] The inlet and outlet of the channel are both located on the cover plate, which facilitates the introduction of solutions or cells, or provides shear stress, and also facilitates chip management.

[0059] The experimental materials and instruments used in this invention were obtained from the following sources:

[0060] The polydimethylsiloxane prepolymer and crosslinking agent (PDMS, Sylgard 184silicone elastomerkit) were both from Dow Corning Incorporated, Inc., USA; the porous polycarbonate membrane was purchased from GVS Corporation, USA; the polyethylene terephthalate porous membrane was from Hangzhou Kebote Filter Material Co., Ltd. (China); and the live cell tracer (CellTracker) was used. Fluorescent Probe, vascular endothelial growth factor (VEGF), human basic fibroblast growth factor (FGF-2), CXCL12, and CXCL8 were purchased from Thermo Fisher Scientific (China); collagen solution (type I rat tail collagen, 4.16 mg / mL) and matrix gel were purchased from Corning Incorporated (USA); laminin and hepatocyte growth factor (HGF) were obtained from Sigma-Aldrich Trading Co., Ltd. (China); human umbilical vein endothelial cells (HUVEC), human liver cancer cells MHCC97-H, and human pancreatic cancer PANC-1 cell lines were provided by the Cell Bank of the Chinese Academy of Sciences (China); human lung adenocarcinoma A549 cells, human breast cancer MDA-MB-231 cells, and human colon cancer HCT116 cell lines were provided by Pronoss (China); DMEM and RPMI were also provided. 1640 medium, fetal bovine serum, and other cell culture-related reagents were purchased from Gibco (USA); silicon wafer molds containing chip cover plates and substrate channel models were manufactured by Suzhou Zhongxin Qiheng Scientific Instruments Co., Ltd. (China); a benchtop spin coater (KW-4A) and a vacuum pump (SC-310V) were purchased from Beijing Saidekais Electronics Co., Ltd.; an electric thermostatic drying oven (DHG-9243B5-Ⅲ, Xinmiao, Shanghai); an inverted biological microscope (ECLIPSE Ts2-FL, Nikon, Japan); an ethylene oxide sterilizer (SQ-H80, Sanqiang Medical Equipment, Henan); a vertical autoclave (LDZF-50L-I, Shenan, Shanghai); a cell culture incubator (Heracell 150i, USA); and a clean bench (SW-CJ-2FD-Ⅱ, Suzhou).

[0061] All other materials not mentioned can be replaced with materials of the same specifications.

[0062] The fabrication method of the microfluidic chip used to simulate the tumor metastasis cascade process, and specific embodiments for simulating and monitoring the tumor metastasis cascade process are as follows:

[0063] Example 1

[0064] like Figure 1 and Figure 2 As shown, this embodiment provides a microfluidic chip for simulating the tumor metastasis cascade process and its fabrication method, with the specific steps as follows:

[0065] (1) Based on standard photolithography, using SU-8 photoresist and Figure 3 The mask shown is fabricated on a clean silicon wafer and contains molds for the chip cover and the substrate channel model, respectively.

[0066] The base mold contains a U-shaped channel model, and the cover mold contains models of the left and right channels.

[0067] (2) Polydimethylsiloxane (PDMS) monomer and crosslinking agent are mixed evenly in a 10:1 ratio, and the mixture is defoamed under vacuum to obtain a PDMS solution. The PDMS solution is poured into the upper and lower chip molds obtained in step (1), and after removing air bubbles using a vacuum pump, it is placed in a 75°C oven to heat and completely cure the PDMS. Then, the PDMS containing the channel structure is cut off from the surface of the mold to obtain the lower chip substrate (e.g., Figure 4 (as shown) and the chip cover plate (such as) Figure 5 (As shown); a manual punch is used to create the liquid inlet and outlet channels at fixed positions on the upper layer of the chip.

[0068] (3) Using uncured PDMS solution as an adhesive, the side of the chip's lower substrate containing the U-shaped channel is bonded to a porous polycarbonate film (10 μm pore size). The PDMS is then cured at 75°C, irreversibly sealing the lower substrate and the porous film together. Using the same method, the side of the chip's upper cover plate containing the left and right channels is then sealed to the middle porous film to obtain a microfluidic chip, such as... Figure 6 and Figure 7 As shown.

[0069] The overall and layered structure of the microfluidic chip prepared above is shown in the following schematic diagram. Figure 1 As shown, it includes a lower chip substrate 1, a middle porous film 2, and an upper chip cover 3; wherein, the lower chip substrate has a U-shaped channel 4, as shown in the figure. Figure 4 As shown, the prepared U-shaped channel has an actual height of 102.6 μm and a width of 1.0 mm; Figure 5 As shown, the upper cover of the chip includes two symmetrical channels, a left channel 5 and a right channel 6. The channels are actually 296.5 μm high and 1.0 mm wide. The left channel 5 overlaps with the left straight inlet channel of the lower U-shaped channel 4, and the right channel 6 overlaps with the right straight outlet channel of the U-shaped channel. The overlap is 5 mm long. The upper and lower channels are separated by a porous membrane in the middle layer. Figure 6 As shown.

[0070] Example 2

[0071] This embodiment provides a microfluidic chip for simulating the tumor metastasis cascade process and its fabrication method, the specific steps of which are as follows:

[0072] (1) Based on standard photolithography, using SU-8 photoresist and Figure 3 The mask shown is used to create a mold containing models of the upper and lower layers of the chip on a clean silicon wafer. The substrate mold contains a model of a U-shaped channel, and the cover mold contains models of the left and right channels.

[0073] (2) Mix the PDMS prepolymer and crosslinking agent evenly in a ratio of 12:1, and obtain a PDMS solution after vacuum defoaming; pour the PDMS solution into the chip substrate and cover plate mold obtained in step (1), remove air bubbles, and place it in a 60℃ oven to heat and completely cure the PDMS. Cut the cured PDMS off the surface of the mold; use a punch to punch the liquid inlet and liquid outlet at the reserved position of the chip cover plate to obtain the upper cover plate and the lower substrate of the chip, respectively.

[0074] (3) Using PDMS solution as an adhesive, the lower chip substrate, upper chip cover and porous polycarbonate film (pore size 12μm) obtained in step (2) are bonded together. The side with channels is bonded to the porous film. The PDMS is cured by heating at 60°C, thereby irreversibly sealing the three together to obtain a microfluidic chip.

[0075] The microfluidic chip prepared above has a U-shaped channel 4 in the lower substrate, which is 80 μm high and 3 mm wide; the upper cover sheet contains two symmetrical channels 5 and 6, which are 350 μm high and 3 mm wide; the left channel 5 overlaps with the left straight inlet channel of the lower U-shaped channel 4, and the right channel 6 overlaps with the right straight outlet channel of the U-shaped channel, with the overlapping part being 10 mm long; the upper and lower channels are separated by a middle porous membrane.

[0076] Example 3

[0077] This embodiment provides a microfluidic chip for simulating the tumor metastasis cascade process and its fabrication method, the specific steps of which are as follows:

[0078] (1) Based on standard photolithography, using SU-8 photoresist and Figure 3 The mask shown is used to create a mold containing the upper and lower channel models of the chip on a clean silicon wafer.

[0079] (2) Mix the PDMS prepolymer and crosslinking agent evenly at a ratio of 15:1, and obtain a PDMS solution after vacuum defoaming; pour the PDMS solution into the upper and lower chip molds obtained in step (1), remove air bubbles, and place them in a 60℃ oven to heat and completely cure the PDMS. Cut the cured PDMS off the surface of the mold; use a punch to punch inlet and outlet ports at the reserved positions on the upper chip layer to obtain the upper chip cover and the lower chip substrate, respectively.

[0080] (3) Using PDMS solution as an adhesive, the lower chip substrate, upper chip cover and porous polycarbonate film (pore size 15μm) obtained in step (2) are bonded together. The side with channels is bonded to the porous film. The PDMS is cured by heating at 60°C, thereby irreversibly sealing the three together to obtain a microfluidic chip.

[0081] The microfluidic chip prepared above has a U-shaped channel 4 in the lower substrate, which is 200 μm high and 2 mm wide; the upper cover sheet of the chip includes two mutually symmetrical left channels 5 and right channels 6, which are 500 μm high and 2 mm wide; the left channel 5 overlaps with the left side of the lower U-shaped channel, and the right channel 6 overlaps with the right side of the U-shaped channel (the overlapping part is 8 mm long), and the upper and lower channels are separated by a middle porous membrane.

[0082] Example 4

[0083] This embodiment provides a microfluidic chip for simulating the tumor metastasis cascade process and its fabrication method, the specific steps of which are as follows:

[0084] (1) Based on standard photolithography, using SU-8 photoresist and Figure 3 The mask shown is used to create a mold containing the upper and lower channel models of the chip on a clean silicon wafer.

[0085] (2) Mix the PDMS prepolymer and crosslinking agent evenly in a ratio of 8:1, and obtain a PDMS polymer solution after vacuum defoaming; pour the PDMS solution into the upper and lower chip molds obtained in step (1), remove air bubbles, and place them in a 75°C oven to heat and completely cure the PDMS. Cut the cured PDMS off the surface of the mold; use a punch to punch inlet and outlet ports at the reserved positions on the upper chip layer to obtain the upper chip cover and the lower chip substrate, respectively.

[0086] (3) Using PDMS solution as an adhesive, the lower chip substrate, the upper chip cover and the polyethylene terephthalate film (8 μm pore size) obtained in step (2) are bonded together. The side with channels is bonded to the porous film. The PDMS is cured by heating at 80°C, thereby irreversibly sealing the three together to obtain a microfluidic chip.

[0087] The microfluidic chip prepared above has a U-shaped channel 4 in the lower substrate, which is 150 μm high and 1 mm wide; the upper cover of the chip includes two mutually symmetrical left channels 5 and right channels 6, which are 400 μm high and 1 mm wide; the left channel 5 overlaps with the left straight inlet channel of the lower U-shaped channel 4, and the right channel 6 overlaps with the right straight outlet channel of the U-shaped channel, with the overlapping part being 3 mm long; the upper and lower channels are separated by a middle porous membrane.

[0088] Example 5

[0089] This embodiment provides a microfluidic chip for simulating the tumor metastasis cascade process and its fabrication method, the specific steps of which are as follows:

[0090] (1) Based on standard photolithography, using SU-8 photoresist and Figure 3 The mask shown is used to create a mold containing the upper and lower channel models of the chip on a clean silicon wafer.

[0091] (2) Mix the PDMS prepolymer and crosslinking agent evenly in a 5:1 ratio, and obtain a PDMS solution after vacuum defoaming; pour the PDMS solution into the upper and lower chip molds obtained in step (1), remove air bubbles, and place them in a 75°C oven to heat and completely cure the PDMS. Cut the cured PDMS off the surface of the mold; use a punch to punch inlet and outlet ports at the reserved positions on the upper chip layer to obtain the upper chip cover and the lower chip substrate, respectively.

[0092] (3) Using PDMS solution as an adhesive, the lower chip substrate, upper chip cover and porous polycarbonate film (pore size 8μm) obtained in step (2) are bonded together. The side with channels is bonded to the porous film. The PDMS is cured by heating at 55°C, thereby irreversibly sealing the three together to obtain a microfluidic chip.

[0093] The microfluidic chip prepared above has a U-shaped channel 4 in the lower substrate, which is 120 μm high and 0.5 mm wide; the upper cover of the chip includes two symmetrical left channels 5 and right channels 6, which are 200 μm high and 0.5 mm wide; the left channel 5 overlaps with the left straight inlet channel of the lower U-shaped channel 4, and the right channel 6 overlaps with the right straight outlet channel of the U-shaped channel, with the overlap portion being 2 mm long; the upper and lower channels are separated by a middle porous membrane.

[0094] Example 6

[0095] This embodiment provides a microfluidic chip for simulating the tumor metastasis cascade process and its fabrication method, the specific steps of which are as follows:

[0096] (1) Based on standard photolithography, using SU-8 photoresist and Figure 3The mask shown is used to create a mold containing the upper and lower channel models of the chip on a clean silicon wafer.

[0097] (2) Mix the PDMS prepolymer and crosslinking agent evenly in a ratio of 13:1, and obtain a PDMS solution after vacuum defoaming; pour the PDMS solution into the upper and lower chip molds obtained in step (1), remove air bubbles, and place them in a 50°C oven to heat and completely cure the PDMS. Cut the cured PDMS off the surface of the mold; use a punch to punch inlet and outlet ports at the reserved positions on the upper chip layer to obtain the upper chip cover and the lower chip substrate, respectively.

[0098] (3) Using PDMS polymer solution as adhesive, the lower chip substrate, upper chip cover and polyethylene terephthalate film (pore size 10μm) obtained in step (2) are bonded together. The side with channels is bonded to the porous film. The PDMS is cured by heating at 55°C, thereby irreversibly sealing the three together to obtain a microfluidic chip.

[0099] The microfluidic chip prepared above has a U-shaped channel 4 in the lower substrate, with a channel height of 150.6 μm and a width of 2 mm; the upper cover of the chip includes two mutually symmetrical left channels 5 and right channels 6 (with a channel height of 352.7 μm and a width of 2 mm). The left channel 5 overlaps with the left straight inlet channel of the lower U-shaped channel 4, and the right channel 6 overlaps with the right straight outlet channel of the U-shaped channel. The upper and lower channels are separated by a middle porous membrane.

[0100] Example 7

[0101] The microfluidic chip prepared above is used to simulate and monitor the cascade process of lung cancer metastasis, as follows: Figure 8 This includes the following steps:

[0102] (1) After rinsing the microfluidic chip prepared in Example 1 with ultrapure water, the entire chip was sterilized using an ethylene oxide sterilizer and then sterilized by irradiation under ultraviolet light. A 0.01 mg / mL type I collagen solution was introduced into the U-shaped channel 4 of the chip substrate after sterilization and incubated in a constant temperature incubator at 37°C for 1 hour. The chip was then flipped over and incubated again to ensure that a thin layer of collagen was evenly coated on the surface of the lower channel.

[0103] (2) Human umbilical vein endothelial cells (HUVECs) were examined using a green fluorescent live cell tracer (such as CellTracker). TM After being marked with Green CMFDA, it is 2×10 5 pcs / cm 2The density of the medium is introduced into the lower U-shaped channel 4 coated with collagen; the chip is flipped over and placed in an incubator and left to stand for 2 hours to allow the endothelial cells to adhere to the wall. Then, endothelial cell culture medium (RPMI 1640 basal medium + 10% fetal bovine serum (FBS) + 1% penicillin-streptomycin (antibiotic, P / S)) is added into the U-shaped channel, and culture is continued for 12 hours until the endothelial cells form tight junctions in the channel.

[0104] (3) Using red fluorescent live cell tracer (CellTracker) TM After labeling human lung cancer A549 cells with Red CMTPX, the A549 cells were resuspended in a matrix gel solution and quickly added to channel 5 on the left side of the upper layer of the chip obtained in step (2). The chip was placed in an incubator and incubated for 15 minutes to allow the matrix gel to solidify. A549 cell-specific culture medium (Ham's F-12K basal medium + 10% FBS + 1% P / S) was added to the channel.

[0105] (4) Subsequently, a high-serum-content culture medium (Ham's F-12K basal medium + 20% FBS) was added to the right channel of the upper layer of the chip obtained in step (3). After the HUVEC cells cultured in the U-shaped channel 4 of the chip obtained in step (3) stabilized and formed tight junctions, a peristaltic pump was used to apply fluid shear stress stimulation to the U-shaped channel 4, adjusting the flow rate to maintain the fluid shear stress level under normal physiological conditions, which is 10 dyn / cm². 2 about.

[0106] (5) The chip is placed in an incubator for culture. The culture medium in the upper channel of the chip is replaced every 24 hours. The migration and location of cancer cells in the chip channel are observed and recorded in real time under a fluorescence microscope every 6 hours. The number of cancer cells that migrate into the lower U-shaped channel and invade the upper right channel of the chip is counted, thereby realizing the simulation and monitoring of the tumor metastasis cascade process.

[0107] Example 8

[0108] The microfluidic chip prepared above is used to simulate and monitor the metastatic cascade process of breast cancer, including the following steps:

[0109] (1) After rinsing the microfluidic chip prepared in Example 2 with ultrapure water, the entire chip was sterilized by high temperature and high pressure steam sterilization and ultraviolet irradiation. A 0.01 mg / mL type I collagen solution was introduced into the U-shaped channel of the lower layer of the chip after sterilization. The chip was placed in a constant temperature incubator at 37°C for 30 min and then incubated again to make the surface of the lower channel uniformly coated with a thin layer of collagen.

[0110] (2) HUVEC cells were used with a green fluorescent live cell tracer (such as CellTracker). TM After being marked with Green CMFDA, it is 2×10 5 pcs / cm 2 The density of the material is introduced into the lower U-shaped channel 4 coated with collagen; the chip is flipped over and placed in an incubator and left to stand for 2 hours to allow the endothelial cells to adhere to the wall. Then, endothelial cell culture medium (RPMI 1640 basal medium + 10% FBS + 1% P / S) is added into the U-shaped channel, and culture is continued for 12 hours until the endothelial cells form tight junctions in the channel.

[0111] (3) Cancer cell spheroids were obtained by pre-culturing human breast cancer MDA-MB-231 cell line and then detected using red fluorescent live cell tracer (CellTracker). TM Red CMTPX) labeled cell spheres. Then, the matrix gel solution resuspending the MDA-MB-231 cell spheres was quickly added to the upper left channel of the chip obtained in step (2), and the chip was placed in an incubator for 10 min to allow the matrix gel to solidify; then MDA-MB-231 cell culture medium (DMEM high glucose basal medium + 10% FBS + 1% P / S) was added to the channel.

[0112] (4) Subsequently, a culture medium containing growth factors (DMEM + 10% FBS + 1% P / S + vascular endothelial growth factor (VEGF, 10-50 ng / mL) + human basic fibroblast growth factor (FGF-2, 10-20 ng / mL)) was added to the right channel of the upper layer of the chip obtained in step (3). Meanwhile, after the HUVECs seeded in the U-shaped channel of the lower layer of the chip have stabilized and formed tight junctions, a peristaltic pump was used to apply fluid shear stress stimulation in the U-shaped channel, adjusting the flow rate to make the fluid shear stress level approximately 15 dyn / cm. 2 .

[0113] (5) Place the chip obtained in step (4) in an incubator for culture. Then, replace the culture medium in the two channels of the upper layer of the chip every 24 hours. Observe and record the migration of breast cancer cells in the chip channels in real time under a fluorescence microscope every 4 hours. Count the number of cancer cells that migrate into the lower U-shaped channel and invade the right channel of the upper layer of the chip, so as to realize the simulation and monitoring of the cascade process of breast cancer cell metastasis.

[0114] Example 9

[0115] In this embodiment, the microfluidic chip prepared above is used to simulate and monitor the metastatic cascade process of liver cancer, including the following steps:

[0116] (1) After rinsing the microfluidic chip obtained in Example 3 with ultrapure water, the entire chip was sterilized by high temperature and high pressure steam sterilization and ultraviolet irradiation. A 0.005 mg / mL laminin solution was introduced into the U-shaped channel of the lower layer of the chip after sterilization. After incubating in a constant temperature incubator at 37°C for 2 hours, the chip was flipped over and incubated again to make the surface of the lower channel uniformly coated with a thin layer of laminin.

[0117] (2) HUVEC cells were used with a green fluorescent live cell tracer (such as CellTracker). TM After being marked with Green CMFDA, it is 2×10 5 pcs / cm 2 The density of the medium is introduced into the lower U-shaped channel 4 coated with the above-mentioned laminin; the chip is flipped and placed in an incubator and left to stand for 4 hours to allow the endothelial cells to adhere to the wall. Then, HUVEC medium (RPMI 1640 basal medium + 10% FBS + 1% P / S) is added into the U-shaped channel, and culture is continued for 24 hours until the endothelial cells form tight junctions in the channel.

[0118] (3) Cancer cell spheroids were obtained by pre-culturing human hepatocellular carcinoma MHCC97-H cell line and then detected using red fluorescent live cell tracer (CellTracker). TM Red CMTPX) labeled cell spheres. Then, the matrix gel solution resuspending MHCC97-H cell spheres was quickly added to the upper left channel of the chip obtained in step (2), the chip was placed in an incubator and incubated for 30 min to solidify the matrix gel, and cell culture medium (DMEM + 10% FBS + 1% P / S) was added to the channel.

[0119] (4) Subsequently, DEME cell culture medium containing hepatocyte growth factor (HGF, 10-20 ng / mL) was added to the right channel of the chip cover obtained in step (3). After the HUVEC cells cultured in the U-shaped channel stabilized and formed tight junctions, a peristaltic pump was used to apply fluid shear stress stimulation within the U-shaped channel, adjusting the flow rate to maintain a fluid shear stress level of approximately 20 dyn / cm. 2 .

[0120] (5) Place the chip obtained in step (4) in an incubator for culture. Then, replace the culture medium in the two channels of the upper layer of the chip every 24 hours. Observe and record the migration of liver cancer cells in the chip channels in real time under a fluorescence microscope every 6 hours. Count the number of cancer cells that migrate into the lower U-shaped channel and invade the right channel of the upper layer of the chip, so as to realize the simulation and monitoring of the cascade process of liver cancer cell metastasis.

[0121] Example 10

[0122] In this embodiment, the microfluidic chip prepared above is used to simulate and monitor the metastatic cascade process of colorectal cancer, including the following steps:

[0123] (1) After rinsing the microfluidic chip obtained in Example 4 with ultrapure water, the entire chip was sterilized by high temperature and high pressure steam sterilization and ultraviolet irradiation. 0.1 mg / mL matrix adhesive solution was introduced into the U-shaped channel of the lower layer of the chip after sterilization. After incubating in a constant temperature incubator at 37°C for 1 hour, the chip was flipped over and incubated again to make the surface of the lower channel uniformly coated with a thin layer of matrix adhesive.

[0124] (2) HUVEC cells were used with a green fluorescent live cell tracer (such as CellTracker). TM After being marked with Green CMFDA, it is 2×10 5 pcs / cm 2 The density of the medium is introduced into the lower U-shaped channel 4 coated with the above-mentioned matrix gel; the chip is flipped and placed in an incubator and left to stand for 6 hours to allow the endothelial cells to adhere to the wall. Then, HUVEC cell culture medium (RPMI 1640 basal medium + 10% FBS + 1% P / S) is added into the U-shaped channel, and culture is continued for 24 hours until the endothelial cells form tight junctions in the channel.

[0125] (3) Cancer cell spheroids were obtained by pre-culturing human colon cancer HCT116 cell line and then detected using red fluorescent live cell tracer (CellTracker). TM Red CMTPX) labeled cell spheres. Then, the matrix gel solution resuspending HCT116 cell spheres was quickly added to the upper left channel of the chip obtained in step (2), and the chip was placed in an incubator for 10 min to solidify the matrix gel. HCT116 cell culture medium (McCoy's 5A + 10% FBS + 1% P / S) was added to the channel.

[0126] (4) Subsequently, HCT116 culture medium containing high concentrations of serum and 10-50 ng / mL VEGF was added to the right channel of the upper layer of the chip obtained in step (3). Meanwhile, after the HUVEC cells cultured in the U-shaped channel stabilized and formed tight junctions, a peristaltic pump was used to apply fluid shear stress stimulation within the U-shaped channel of the chip, adjusting the flow rate to maintain a fluid shear stress level of approximately 30 dyn / cm. 2 .

[0127] (5) Place the chip obtained in step (4) in an incubator for culture. Then, replace the culture medium in the two channels of the upper layer of the chip every 24 hours. Observe and record the migration of colon cancer cells in the chip channels in real time under a fluorescence microscope every 4 hours. Count the number of cancer cells that migrate into the lower U-shaped channel and invade the right channel of the upper layer of the chip, so as to realize the simulation and monitoring of the cascade process of colon cancer cell metastasis.

[0128] Example 11

[0129] In this embodiment, the microfluidic chip prepared above is used to simulate and monitor the metastatic cascade process of pancreatic cancer, including the following steps:

[0130] (1) After rinsing the microfluidic chip obtained in Example 5 with ultrapure water, the entire chip was sterilized by high temperature and high pressure steam sterilization and ultraviolet irradiation. A 0.005 mg / mL laminin solution was introduced into the U-shaped channel of the lower layer of the chip after sterilization. The chip was placed in a constant temperature incubator at 37°C for 30 min and then incubated again to make the surface of the lower channel uniformly coated with a thin layer of laminin.

[0131] (2) HUVEC cells were used with a green fluorescent live cell tracer (such as CellTracker). TM After being marked with Green CMFDA, it is 2×10 5 pcs / cm 2 The density of the medium is introduced into the lower U-shaped channel 4 coated with the above-mentioned laminin; the chip is flipped and placed in an incubator and left to stand for 8 hours to allow the endothelial cells to adhere to the wall. Then, HUVEC cell culture medium (RPMI 1640 basal medium + 10% FBS + 1% P / S) is added into the U-shaped channel, and culture is continued for 20 hours until the endothelial cells form tight junctions in the channel.

[0132] (3) Cancer cell spheroids were obtained by pre-culturing human pancreatic cancer PANC-1 cell line and detected using red fluorescent live cell tracer (CellTracker). TM Red CMTPX) labeled cell spheres. Then, the matrix gel solution resuspending the PANC-1 cell spheres was quickly added to the upper left channel of the chip obtained in step (2), and the chip was placed in an incubator for 25 min to allow the matrix gel to solidify. PANC-1 cell culture medium (DMEM high glucose basal medium + 10% FBS + 1% P / S) was added to the channel.

[0133] (4) Subsequently, PANC-1 cell culture medium containing 50-100 ng / mL CXCL12 and 10-20 ng / mL CXCL8 was added to the right channel of the upper layer of the chip obtained in step (3). After the HUVECs in the U-shaped channel of the chip in step (3) have stabilized and formed tight junctions, a peristaltic pump was used to apply fluid shear stress stimulation within the U-shaped channel, adjusting the flow rate to make the fluid shear stress level approximately 40 dyn / cm. 2 .

[0134] (5) Place the chip obtained in step (4) in an incubator for culture. Then, replace the culture medium in the two channels of the upper layer of the chip every 36 hours. Observe and record the migration of pancreatic cancer cells in the chip channels in real time under a fluorescence microscope every 4 hours. Count the number of cancer cells that migrate into the lower U-shaped channel and invade the right channel of the upper layer of the chip, so as to realize the simulation and monitoring of the pancreatic cancer cell metastasis cascade process.

[0135] Example 12

[0136] In this embodiment, the microfluidic chip prepared above is used to simulate and monitor the cascade process of lung cancer metastasis, including the following steps:

[0137] (1) After rinsing the microfluidic chip obtained in Example 6 with ultrapure water, the entire chip was sterilized by high temperature and high pressure steam sterilization and ultraviolet irradiation. A solution containing 10 μg / mL fibronectin, 0.1% gelatin and 0.1 mg / mL polylysine was introduced into the U-shaped channel of the lower layer of the chip after sterilization. After incubating in a 37°C incubator for 1 h, the chip was flipped and incubated again to make the matrix protein uniformly coated on the surface of the lower channel.

[0138] (2) HUVEC cells were used with a green fluorescent live cell tracer (such as CellTracker). TM After being marked with Green CMFDA, it is 2×10 5 pcs / cm 2 The density of the medium is introduced into the lower U-shaped channel 4 coated with the matrix protein; the chip is flipped and placed in an incubator and left to stand for 8 hours to allow the endothelial cells to adhere to the wall. Then, HUVEC cell culture medium (RPMI 1640 basal medium + 10% FBS + 1% P / S) is added into the U-shaped channel, and culture is continued for 20 hours until the endothelial cells form tight junctions in the channel.

[0139] (3) Using red fluorescent live cell tracer (CellTracker) TM After labeling human lung cancer A549 cells with Red CMTPX, the A549 cells were resuspended in a matrix gel solution and quickly added to channel 5 on the left side of the upper layer of the chip obtained in step (2). The chip was placed in an incubator and incubated for 20 minutes to allow the matrix gel to solidify. A549 cell-specific culture medium (Ham's F-12K + 10% FBS + 1% P / S) was added to the channel.

[0140] (4) Subsequently, high serum content culture medium, Ham's F-12K basal medium + 20% FBS, was added to the right channel of the upper layer of the chip obtained in step (3). After the seeded HUVEC cells stabilized and formed tight junctions, a peristaltic pump was used to apply fluid shear stress stimulation within the U-shaped channel of the chip, adjusting the flow rate to maintain a fluid shear stress level of approximately 20 dyn / cm. 2 .

[0141] (5) Place the chip obtained in step (4) in an incubator for culture. Then, replace the culture medium in the two channels of the upper layer of the chip every 24 hours. Observe and record the migration of lung cancer cells in the chip channels in real time under a fluorescence microscope every 6 hours. Count the number of cancer cells that migrate into the lower U-shaped channel and invade the right channel of the upper layer of the chip, so as to realize the simulation and monitoring of the lung cancer cell metastasis cascade process.

[0142] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any non-essential changes, modifications, substitutions, combinations, simplifications, or adjustments made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall fall within the protection scope of the present invention.

Claims

1. A microfluidic chip simulating the cascade process of tumor metastasis, characterized in that, From bottom to top, the structure includes: a substrate (1), a middle porous membrane (2), and a cover plate (3); a U-shaped channel (4) is provided on the side of the substrate (1) where the middle porous membrane (2) is attached, and a left channel (5) and a right channel (6) are provided on the side of the cover plate (3) where the middle porous membrane (2) is attached; the substrate (1), the middle porous membrane (2), and the cover plate (3) are attached together, the left channel (5) overlaps with the left channel of the U-shaped channel (4), and the right channel (6) overlaps with the right channel of the U-shaped channel (4); the left channel (5) is used for the culture of tumor cells, the right channel (6) is used to provide space for the extravasation and colonization of tumor cells, and the U-shaped channel (4) is used to simulate the vascular structure and fluid environment, providing environmental conditions for the infiltration, circulation, and extravasation of tumor cells; the tumor cells in the left channel (5) can migrate through the channel and the porous membrane in the U-shaped channel (4) and the right channel (6).

2. The microfluidic chip for simulating the tumor metastasis cascade process according to claim 1, characterized in that, The middle porous film (2) is made of porous polycarbonate or polyethylene terephthalate, and micropores with a pore size between 5 and 15 μm are uniformly distributed on the middle porous film (2).

3. The microfluidic chip for simulating the tumor metastasis cascade process according to claim 1 or 2, characterized in that, The substrate (1) and cover plate (3) are made of light-transmitting polydimethylsiloxane.

4. The microfluidic chip for simulating the tumor metastasis cascade process according to claim 3, characterized in that, The height of the U-shaped channel (4) is 80-300 μm and the width is 0.5-3 mm; the height of the left channel (5) is 200-500 μm and the width is 0.5-3 mm; the dimensions of the right channel (6) are the same as those of the left channel (5); the length of the overlap between the left channel (5) and the right channel (6) and the U-shaped channel (4) is 2-10 mm.

5. The microfluidic chip for simulating the tumor metastasis cascade process according to claim 4, characterized in that, The U-shaped channel (4) has an inlet (41) and an outlet (42) at both ends. The left channel (5) has an inlet (51) and an outlet (52) at both ends. The right channel (6) has an inlet (61) and an outlet (62) at both ends. The inlet (41) and outlet (42), the inlet (51) and outlet (52), and the inlet (61) and outlet (62) are all located on the cover plate (3).

6. A method for fabricating a microfluidic chip simulating a tumor metastasis cascade process, characterized in that, Includes the following steps: Microfluidic chip mold fabrication based on standard photolithography: The substrate and the cover plate mold are fabricated on a clean silicon wafer using SU-8 photoresist. The substrate mold contains a model of a U-shaped channel (4), and the cover plate mold contains a model of a left channel (5) and a right channel (6). A polydimethylsiloxane solution was prepared and poured into the base mold and the cover plate mold, respectively, and then heated to cure. Cut the base and cover plate with channels from the mold surface, and use a manual punch to make liquid inlet (41) and liquid outlet (42), liquid inlet (51) and liquid outlet (52), and liquid inlet (61) and liquid outlet (62) at the corresponding positions on the cover plate; By using plasma treatment or polydimethylsiloxane solution as an adhesive, the side of the substrate with the U-shaped channel is sealed together with the middle porous film, and then the other side of the middle porous film is bonded to the cover plate to obtain the microfluidic chip simulating the tumor metastasis cascade process.

7. The method for fabricating a microfluidic chip simulating a tumor metastasis cascade process according to claim 6, characterized in that, The preparation of the polydimethylsiloxane solution involves adding a crosslinking agent to the polydimethylsiloxane monomer, wherein the mass ratio of the polydimethylsiloxane monomer to the crosslinking agent is 5 to 15:

1.

8. The method for fabricating a microfluidic chip simulating a tumor metastasis cascade process according to claims 6 and 7, characterized in that, The temperature for heat curing is 50–80°C.

9. A method for simulating and monitoring the cascade process of tumor metastasis using a microfluidic chip, characterized in that, Includes the following steps: After cleaning and sterilizing the microfluidic chip, a matrix protein solution is introduced into the U-shaped channel (4). The microfluidic chip is placed in a constant temperature incubator at 37°C and incubated for 10 min to 2 h. Then, the microfluidic chip is flipped over and incubated again to make the surface of the U-shaped channel (4) uniformly coated with a thin layer of matrix protein. A suspension of vascular endothelial cells labeled with a live cell tracer was introduced into the U-shaped channel (4) coated with the matrix protein. The microfluidic chip was flipped and placed in an incubator to stand for 2 to 12 hours, allowing the cells to adhere to the walls of the U-shaped channel (4). The U-shaped channel (4) was then filled with vascular endothelial cell culture medium, and cultured for another 6 to 24 hours to allow the vascular endothelial cells to stabilize and form tight junctions. A matrix gel solution of resuspended tumor cells labeled with live cell tracer was introduced into the left channel (5). The microfluidic chip was placed in a 37°C incubator and incubated for 10-30 minutes to solidify the matrix gel of the resuspended tumor cells. Tumor cell culture medium was added into the left channel (5). A special culture medium containing biomolecules that can affect the migration of tumor cells is added into the right channel (6); a peristaltic pump is used to apply fluid shear stress stimulation in the U-shaped channel (4); The microfluidic chip treated above was placed in an incubator for culture. The tumor cell culture medium in the left channel (5) was replaced every 12 to 48 hours. The migration and location of tumor cells in the microfluidic chip channel were observed and recorded in real time under a fluorescence microscope every 2 to 12 hours. The number of tumor cells that migrated into the U-shaped channel (4) and invaded the right channel (6) was counted to simulate and monitor the tumor metastasis cascade process.

10. The method for simulating and monitoring the tumor metastasis cascade process using a microfluidic chip according to claim 9, characterized in that: The special culture medium contains biomolecules that can affect the migration of tumor cells, including high concentrations of serum, cytokines, growth factors and / or chemokines.

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