CN104830683B - A kind of bionical micro-fluidic chip for simulating interior tumor cell and its transfer microenvironment - Google Patents

Abstract

The invention discloses a bionic microfluidic chip for simulating in vivo tumor cells and their transfer microenvironment. The microfluidic chip is composed of three layers of PDMS substrates and two layers of porous PDMS membranes interlaced and irreversibly sealed. The first layer of PDMS substrate is provided with channels for air circulation, inlet and outlet for liquid in and out, and the upper part of the vacuum channel; the second layer of PDMS substrate is provided with channels for liquid circulation, inlet and outlet for liquid in and out , the upper part of the vacuum channel; the third layer of PDMS substrate is provided with a cell culture chamber for cell culture; the channel on the first layer of PDMS substrate is located at the upstream of the channel on the second layer of PDMS substrate, and the third layer of PDMS substrate The cell culture chamber is located downstream of the channel on the second layer of PDMS substrate. The microfluidic chip of the present invention can be used to detect the process of tumor cell metastasis in vitro, and provide a basis for clinically formulating tumor treatment plans.

Description

A biomimetic microfluidic device for simulating in vivo tumor cells and their metastatic microenvironment chip

technical field

The invention belongs to the field of clinical application, and relates to a bionic microfluidic chip for simulating the microenvironment of tumors and their metastases in the body. The microfluidic chip of the present invention can dynamically monitor the metastasis process of tumor cells, define the migration mode of tumor cells, and provide guidance for preventing and treating tumor metastasis.

Background technique

Cancer cell metastasis is the main cause of death from all cancers, including lung cancer, and is one of the biggest challenges for basic researchers and clinical scholars. Cancer cell metastasis is a complex physiological process that usually includes primary site tumor lesion proliferation, tumor cell detachment, tumor cell migration, extravasation, and formation of metastatic tumor cell clusters at second organs. Tumor cells with a metastatic phenotype exhibit the following properties: enhanced cell mobility, increased ability to degrade basement membrane components, increased ability to migrate to surrounding cells, increased ability to penetrate lymphatic or blood vessels, The ability to self-proliferate is enhanced. However, tumor cell migration is highly organ-selective, a process that involves interactions between tumor cells and host organs. So far, the precise mechanism of organ-specific tumor metastasis has not been fully understood.

Lung cancer is the leading cause of cancer death in the world, and the metastasis of cancer cells to distant organs is the main cause of lung cancer death. Clinical results show that lung cancer often metastasizes to brain tissue, bone tissue and liver tissue. A better understanding of the metastatic patterns of lung cancer is critical for formulating treatment strategies for lung cancer patients. Therefore, it is an urgent problem to develop an in vitro cell culture model that can simulate the in vivo microenvironment of lung cancer metastasis.

Understanding the pathology of lung cancer cell metastasis requires studying the function of physiologically active cancer cells and tissues in the context of the entire physiologically active lung and distant organs. However, currently available animal models for evaluating normal physiological and disease processes are expensive, subject to lengthy experimental cycles, and present various ethical controversies. More importantly, the above-mentioned animal models cannot well control the localization and tropism of the metastatic lung cancer cells, so that they cannot correctly reflect the physiological state of lung cancer cell metastasis in humans. In recent years, an in vitro 3D cell culture model has become more widely used. The above-mentioned in vitro 3D cell culture model has been used for the co-culture of various tumor cells and mesenchymal cells. These models allow easy manipulation of conditions to study the effects of paracrine signaling on different cell types. The inventors upgraded and modified the existing 3D cell culture model, and developed a bionic microfluidic chip that can be used to simulate the microenvironment of lung cancer metastasis in vivo.

Contents of the invention

In order to solve the problems existing in the prior art, the present invention provides a bionic microfluidic chip for simulating in vivo tumor cells and their metastatic microenvironment. In this microfluidic chip, different cells can be co-cultured to dynamically monitor the transfer process of upstream tumor cells to downstream target organs, providing a basis for clinically formulating tumor treatment plans.

The above-mentioned microfluidic chip of the present invention adopts the following design scheme:

The microfluidic chip is an airtight whole formed by interlacing and irreversibly sealing three layers of PDMS substrates and two layers of porous PDMS membranes; the first layer of PDMS substrate 1 is provided with air channels 11 for air circulation, The first layer of liquid inlet 12 and the first layer of liquid outlet 13 for liquid to enter and exit are respectively located on the upper half of the first vacuum channel 14 and the second vacuum channel 15 on both sides of the air channel 11; the second layer of PDMS substrate 2 is provided with a liquid channel 21 for liquid circulation, a second-layer liquid inlet 22 and a second-layer liquid outlet 23 for liquid to enter and exit, and the first vacuum channel 14 and the second vacuum channel respectively located on both sides of the liquid channel 21 15, the side of the liquid channel 21 is provided with three connecting channels extending outward, which are respectively the first connecting channel 211, the second connecting channel 212, and the third connecting channel 213. The first connecting channel The ends of the channel 211, the second connecting channel 212, and the third connecting channel 213 are respectively provided with a first inlet 214, a second inlet 215, and a third inlet 216; The cultivated first cell culture chamber 31, the second cell culture chamber 32, and the third cell culture chamber 33; the first cell culture chamber 31, the second cell culture chamber 32, and the third cell culture chamber 33 respectively Located below the first connecting channel 211, the second connecting channel 212, and the third connecting channel 213, through the first porous PDMS membrane 5 and the first connecting channel 211, the second connecting channel 212, the third connecting channel 213 communicates; the upper half of the first vacuum channel 14 and the second vacuum channel 15 are formed correspondingly to the lower half of the first vacuum channel 14 and the second vacuum channel 15 The first vacuum channel 14 and the vacuum channel 15 with a complete structure; the first layer of liquid inlet 12 and the first layer of liquid outlet 13 are respectively arranged upstream and downstream of the air channel 11 and on the same side; The second-layer liquid inlet 22 and the second-layer liquid outlet 23 are arranged upstream, downstream and on the same side of the liquid channel 21, the air channel 11 is located directly above the liquid channel 21, and the air channel 11 The opening of the liquid channel 21 is opposite to the opening of the liquid channel 21 and separated by the second porous PDMS membrane 4; the air channel 11 covers the upstream end of the liquid channel 21, the first connecting channel 211, the second connecting channel 211 The second connecting channel 212 and the third connecting channel 213 are located at the downstream end of the liquid channel 21 .

Further, the first porous PDMS membrane 5 and the second porous PDMS membrane 4 have a thickness of 10 μm and a pore diameter of 10 μm.

Further, in a specific embodiment of the present invention, the cross section of the air passage 11 and the liquid passage 21 is rectangular, and the dimensions are: length 10mm×width 4mm, length 35mm×width 4mm; the first vacuum passage 14 1. The cross-section of the second vacuum channel 15 is rectangular, and its size is: 7 mm high and 2 mm wide; the cross-sections of the first cell culture chamber 31, the second cell culture chamber 32, and the third cell culture chamber 33 The shape is rectangular, and the dimensions are: height 1.5mm, width 1.5mm.

Further, in a specific embodiment of the present invention, the length of the air channel 11 is 10mm; the length of the liquid channel 21 is 35mm; the first connecting channel 211, the second connecting channel 212, the second connecting channel The distance between the three connecting channels 213 and one end of the liquid channel 21 is 2.2 mm.

Further, in a specific embodiment of the present invention, the distance between the first layer of liquid inlet 12 and the first layer of liquid outlet 13 is 15 mm; the second layer of liquid inlet 22 and the second layer of liquid outlet 23 The distance is 15mm.

Further, in a specific embodiment of the present invention, the first connecting channel 211 , the second connecting channel 212 , and the third connecting channel 213 are parallel to each other.

Further, in a specific embodiment of the present invention, the distance between the first vacuum channel 14 and the air channel 11 is 2 mm; the distance between the second vacuum channel 15 and the air channel 11 is 2 mm.

All the channels on the microfluidic chip of the present invention are grooves arranged on the substrate; the cell culture chambers on the microfluidic chip of the present invention are grooves arranged on the substrate.

The materials constituting the upper and lower substrates of the microfluidic chip can be PDMS (polydimethylsiloxane), PMMA (polymethyl methacrylate), PC (polycarbonate), COC resin, ABS (acrylonitrile- styrene-butadiene copolymer), glass, quartz, or copper. In a specific embodiment of the present invention, the preparation material of the substrate 1 and the substrate 2 is PDMS.

The present invention also provides a preparation method for the above-mentioned microfluidic chip, the preparation method comprising the following steps:

(1) Prepare the SU-8 positive mold with the microchannel and microstructure in the above-mentioned microfluidic chip;

(2) Take the SU-8 positive mold prepared in step (1) as a template, and use PDMS as a raw material copy to prepare a first layer of PDMS substrate, a second layer of PDMS substrate, and a third layer of PDMS substrate;

(3) Prepare two porous PDMS membranes in the above-mentioned microfluidic chip;

(4) the first layer of PDMS substrate prepared in step (2), the second layer of PDMS substrate, the third layer of PDMS substrate and the two porous PDMS membranes prepared in step (3) It is formed by interlacing and sealing each other.

Further, in a specific embodiment of the present invention, the preparation method of the microfluidic chip of the present invention is as follows:

(1) Use computer-aided design software CAD to draw the microchannel and microstructure diagram in the above-mentioned microfluidic chip; print the diagram on SU-8 film (Microchem, model 2075) as a mask, and make it by standard photolithography process Mold, standard photolithography process is well known to those skilled in the art;

(2) Mix PDMS (Dow Corning, product number: 0007883528) and curing agent at a mass ratio of 10:1, pour the mold on the surface of the mold prepared in step (1) after vacuuming in a vacuum drying oven, and bake at 80°C for 1 hour;

(3) Slowly tear off the PDMS on the template after cooling, drill the inlet and outlet at the corresponding position of the PDMS substrate, and then cut it into a suitable size;

(4) The porous PDMS membrane is made by mixing PDMS and curing agent at a mass ratio of 15:1, using a homogenizer at 3000 rpm for 1 minute, vacuuming, pouring it on the surface of the mold prepared in step (1), and baking at 65°C overnight; After cooling, slowly tear off the PDMS on the template and set it aside.

Su-8 photolithography technology: The new chemically amplified negative image SU-8 photoresist overcomes the problem of insufficient aspect ratio of ordinary photoresists using UV lithography, and is very suitable for the preparation of high aspect ratio microstructures, so SU-8 Glue 8 is a negative-working, epoxy-based, near-ultraviolet photoresist. It has very low light absorption in the near ultraviolet (365nm-400nm) range, and the exposure amount obtained by the entire photoresist layer is uniform, and thick film patterns with vertical side walls and high aspect ratio can be obtained; it also has Good mechanical properties, chemical resistance and thermal stability; SU-8 is cross-linked after being exposed to ultraviolet radiation, it is a chemically expanded negative gel, which can form complex structures such as steps; It is conductive and can be directly used as an insulator during electroplating. Due to its many advantages, SU-8 glue is being gradually applied in the fields of MFMS, chip packaging and micromachining. It is a new technology in the field of micromachining to directly use SU-8 photoresist to prepare microstructures and microparts with high aspect ratio.

The present invention also provides the application of the above-mentioned microfluidic chip in monitoring the process of tumor cell metastasis. The operation steps of using the microfluidic chip of the present invention to monitor the process of tumor cell metastasis in vivo are as follows:

1. Substantial simulation: Epithelial cells and tumor cells are co-cultured on the side of the porous PDMS membrane 4 in the microfluidic chip that contacts the air.

2. Interstitial simulation: co-cultivate vascular endothelial cells and interstitial cells (such as fibroblasts and monocytes) on the side of the porous PDMS membrane 4 in the microfluidic chip that contacts the liquid.

3. Simulation of tumor cell metastasis target organs: brain tissue cells, bone tissue cells, and liver tissue cells are respectively cultured in 3D in the cell culture chambers 31 , 32 , and 33 in the microfluidic chip.

4. To test the effectiveness of the tumor metastasis microenvironment constructed in vitro

By detecting cell viability, the tightness of the connection between vascular endothelial cells and epithelial cells, and the expression of cancer-related fibroblasts and macrophage-specific proteins induced by tumor cells, the simulated tumor cell metastasis constructed by the microfluidic chip of the present invention is evaluated. Effectiveness of biomimetic models of microenvironments.

5. Detection of tumor cell metastasis process

Evaluate the generation of metastatic tumor cells by detecting the expression of epithelial-mesenchymal transition (EMT) marker proteins in tumor cells; compare the growth pattern of metastatic tumor cells by observing the cell morphology; mark the tumor cells with mesenchymal-epithelial transition (MET) Protein expression evaluates changes in metastatic tumor cells after migration to target organs; evaluates whether metastatic tumor cells have invaded target organs by detecting the expression of target organ damage marker proteins.

In a specific embodiment of the present invention, the epithelial cells are bronchial epithelial cells; the tumor cells are lung cancer cells, that is, a bionic model simulating the metastatic microenvironment of lung cancer cells is constructed by using the microfluidic chip of the present invention.

Principle: The present invention adopts PDMS and permeable membrane material with meshes, and designs and manufactures a cell-to-cell, cell-to-culture medium, tissue-to-tissue, organ-to-microenvironment interaction characteristics and principles of fluid mechanics in the body. A high-throughput microfluidic laboratory-on-a-chip that can approach the anatomical structure of the lung and simulate the physiological function of the lung with multi-unit integration and multi-channel connection. The core issue of the laboratory design is how to reconstruct the anatomical structure of the lung, including the lung parenchyma and interstitium including bronchi and alveoli at all levels, and how to simulate the main physiological function of the lung, namely gas exchange. A permeable membrane with a mesh is used to replace the elastic fibers that can promote the retraction of the expanded alveoli during breathing, and two-dimensional culture of bronchial epithelial cells, vascular endothelial cells, macrophages, and fibroblasts on the upper and lower sides of the membrane , simulating the lung parenchyma and interstitium, and constitutes an air-blood barrier, and then in the direction parallel to the membrane, gas and liquid are respectively introduced to supply oxygen and nutrients, and two vacuum channels are connected on both sides perpendicular to the membrane, simulating the human body Breathing rhythm. When gas is pumped into the vacuum channel, the permeable membrane retracts, oxygen is pumped into the gas channel, and the alveoli expand, simulating the inspiratory function of the lungs; when the vacuum channel pumps gas back, the permeable membrane stretches, oxygen is pumped out of the gas channel, and the alveoli contract. Simulates lung expiratory function. So far, a bionic lung chip physiological model close to the anatomical structure of the lung and capable of simulating the main physiological functions of the lung has been constructed. On the basis of the above-mentioned bionic lung physiological model of the chip, and according to the fact that most lung cancers originate from the bronchial mucosal epithelium, the lung cancer was inoculated in the bronchial epithelial cell area respectively to construct the bionic lung cancer pathological model of the chip; Glial cell culture room, bone cell culture room, and liver cell culture room were built on the pathological model to simulate the most frequently metastatic target organs of lung cancer, such as the brain, bone, and liver, to construct pathological models of lung cancer at different stages of metastasis, and to reproduce the full range of invasion and metastasis of lung cancer. process.

Advantage of the present invention and beneficial effect are:

(1) The microfluidic chip of the present invention can realize three-dimensional culture of cells to better complete the study of biological functions in vivo; the reagent consumption of the microfluidic chip for detection is significantly lower than that of traditional platforms.

(2) Cell culture in the microfluidic chip platform of the present invention has good adaptability; the main material of the microfluidic chip is PDMS, and PDMS has good biocompatibility and high gas permeability, and gas can pass through PDMS smoothly , so as to ensure the gas exchange between the cells cultured in the chip and the foreign medium.

(4) The static macro-environment of cell growth on the traditional platform is very different from the tiny three-dimensional space in which cells live. The microfluidic chip system makes up for the shortcomings of the traditional platform in this regard. It has better airtightness, so the cell living space The size of the cell is more similar to the physiological microspace in which cells live in the human body, and the in-situ online detection in this tiny space further increases the reliability of the detection results.

(5) The microfluidic chip platform of the present invention is easy to operate and saves time and effort.

Description of drawings

Fig. 1 shows the exploded view of the microfluidic chip structure of the present invention;

Fig. 2 shows the exploded view of the microfluidic chip structure of the present invention;

Fig. 3 shows the top view of the overall structure of the microfluidic chip of the present invention;

Fig. 4 shows the side view of the first layer substrate structure of the microfluidic chip of the present invention;

Fig. 5 shows the side view of the second layer substrate structure of the microfluidic chip of the present invention;

Fig. 6 shows the side view of the third layer substrate structure of the microfluidic chip of the present invention;

Fig. 7 shows the side view of the porous PDMS membrane of the microfluidic chip of the present invention;

Figure 8 shows the detection of cell viability in the chip by H33342/PI staining and the connection of bronchial epithelial cells 16HBE and vascular endothelial cells HUVEC by immunostaining, wherein, A: cell morphology of 16HBE cells after 24H; B: cell morphology of HUVEC cells after 24H Cell morphology; C: Cell morphology of 16HBE cells after 48H; D: Cell morphology of HUVEC cells after 48H; E: H33342/PI staining of 16HBE cells: F: H33342/PI staining of HUVEC cells; G: E-cadherin protein in 16HBE cells Immunostaining; H: Immunostaining of E-cadherin protein in HUVEC cells;

Figure 9 shows the colocalization of lung cancer cell A549 and bronchial epithelial cells 16HBE detected by Immunofluorescence cell tracker expression assay and the expression of specific proteins of lung cancer cells, fibroblasts and macrophages detected by immunostaining, wherein, A: 16HBE Cell morphology, B: cell morphology of 16HBE co-cultured with A549; C: cell tracker staining of 16HBE cells; D: cell tracker staining of 16HBE and A549 co-cultured; E: CEA protein immunostaining of 16HBE cells; F: CEA co-cultured with 16HBE and A549 Protein immunostaining; G: α-SMA protein immunostaining in fibroblasts; H: α-SMA protein immunostaining in CAF (cancer-associated fibroblasts); I: CD206 protein immunostaining in macrophages; J: CAM ( CD206 protein immunostaining in cancer-associated macrophages);

Figure 10 shows the detection of the expression of epithelial-mesenchymal transition markers in lung cancer cells by immunostaining, wherein, A: cell immunostaining map; B: optical density value statistical map;

Figure 11 shows the migration of lung cancer cells A549 to the distal tissue and the growth pattern at the distal tissue, wherein, A: the statistics of the number of lung cancer cells migrating; B: the cell morphology of lung cancer cells at the distal tissue;

Figure 12 shows the use of immunostaining to detect the expression of characteristic proteins in distal tissues, wherein, A: immunostaining diagram of characteristic proteins; B: statistical diagram of optical density values;

Among them, 1: first layer substrate; 11: air channel; 12: first layer liquid inlet; 13: first layer liquid outlet; 14: first vacuum channel; 15: second vacuum channel; 2: second layer Substrate; 21: liquid channel; 22: second layer liquid inlet; 23: second layer liquid outlet; 211: first connection channel; 212: second connection channel; 213: third connection channel; 214: first inlet ; 215: second entrance; 216: third entrance; 3: third substrate; 31: first cell culture chamber; 32: second cell culture chamber 33: third cell culture chamber; 4: second porous PDMS membrane; 5: first porous PDMS membrane.

detailed description

The content of the present invention can be understood more easily by referring to the following examples, which are only for further illustrating the present invention, and are not meant to limit the scope of the present invention.

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

Example 1 A bionic microfluidic chip for simulating the microenvironment of lung cancer metastasis in vivo

The microfluidic chip of the present invention is an airtight whole formed by interlacing and irreversible sealing of three layers of PDMS substrates and two layers of porous PDMS membranes; the first layer of PDMS substrate 1 is provided with an air channel 11 for air circulation , the first layer of liquid inlet 12 and the first layer of liquid outlet 13 for liquid to enter and exit, respectively located in the upper half of the first vacuum channel 14 and the second vacuum channel 15 on both sides of the air channel 11; the second layer of PDMS substrate 2 A liquid passage 21 for liquid circulation, a second-layer liquid inlet 22 and a second-layer liquid outlet 23 for liquid to flow in and out are arranged on the top, and are respectively located under the first vacuum passage 14 and the second-layer liquid 15 on both sides of the liquid passage 21. In the half part, there are three connecting channels extending outward on the side of the liquid channel 21, which are respectively the first connecting channel 211, the second connecting channel 212, the third connecting channel 213, the first connecting channel 211, and the second connecting channel. 212, the end of the third connection channel 213 is respectively provided with a first inlet 214, a second inlet 215, a third inlet 216; the third layer of PDMS substrate 3 is provided with a first cell culture chamber 31 for cell culture, a second The cell culture chamber 32, the third cell culture chamber 33; the first cell culture chamber 31, the second cell culture chamber 32, and the third cell culture chamber 33 are respectively located in the first connecting channel 211, the second connecting channel 212, and the third connecting channel 213, communicate with the first connecting channel 211, the second connecting channel 212, and the third connecting channel 213 through the first porous PDMS membrane 5; the upper part of the first vacuum channel 14, the second vacuum channel 15 and the first The structure of the lower part of the vacuum channel 14 and the second vacuum channel 15 corresponds to the first vacuum channel 14 and the second vacuum channel 15; the first layer of liquid inlet 12 and the second layer of liquid outlet 13 are respectively arranged in the air channel 11 upstream and downstream and on the same side; the second layer of liquid inlet 22 and the second layer of liquid outlet 23 are arranged on the upstream and downstream of the liquid channel 21 and on the same side, the air channel 11 is located directly above the liquid channel 21, the opening of the air channel 11 and The opening of the liquid channel 21 is opposite and separated by the second porous membrane 4; the air channel 11 covers the upstream end of the liquid channel 21, the first connecting channel 211, the second connecting channel 212, and the third connecting channel 213 are located downstream end.

The second porous PDMS membrane 4 and the first porous PDMS membrane 5 have a thickness of 10 μm and a pore diameter of 10 μm.

The cross section of the air channel 11 and the liquid channel 21 is rectangular, and the size is: length 10mm×width 4mm, length 35mm×width 4mm; the cross section of the first vacuum channel 14 and the second vacuum channel 15 is rectangular, and the size is: height 7mm , width 2mm; the cross-sectional shape of the first cell culture chamber 31, the second cell culture chamber 32, and the third cell culture chamber 33 is rectangular, and the dimensions are: height 1.5mm, width 1.5mm.

The length of the air channel 11 is 10 mm; the length of the liquid channel 21 is 35 mm; the distance between the first connecting channel 211 , the second connecting channel 212 and the third connecting channel 213 and one end of the liquid channel 21 is 2.2 mm.

The distance between the first-layer liquid inlet 12 and the first-layer liquid outlet 13 is 15 mm; the distance between the second-layer liquid inlet 22 and the second-layer liquid outlet 23 is 15 mm.

The first connecting channel 211 , the second connecting channel 212 and the third connecting channel 213 are parallel to each other.

The distance between the first vacuum channel 14 and the air channel 11 is 2 mm; the distance between the second vacuum channel 15 and the air channel 11 is 2 mm.

All the channels on the microfluidic chip of the present invention are grooves arranged on the substrate; the cell culture chambers on the microfluidic chip of the present invention are grooves arranged on the substrate.

Preparation of the microfluidic chip in Example 2 Example 1

The preparation method of the microfluidic chip in embodiment 1 comprises the following steps:

(1) Use computer-aided design software CAD to draw the microchannel and microstructure diagram in the above-mentioned microfluidic chip; print the diagram on SU-8 film (Microchem, model 2075) as a mask, and make it by standard photolithography process Mold, standard photolithography process is well known to those skilled in the art;

(2) Mix PDMS (Dow Corning, article number: 0007883528) and curing agent at a mass ratio of 10:1, and then pour it on the surface of the mold prepared in step (1) after vacuuming in a vacuum drying oven, and bake at 80°C for 1 hour;

(3) Slowly tear off the PDMS on the template after cooling, drill the inlet and outlet at the corresponding position of the PDMS substrate, and then cut it into a suitable size;

(4) The porous PDMS membrane is made by mixing PDMS and curing agent at a mass ratio of 15:1, using a homogenizer at 3000 rpm for 1 minute, vacuuming, pouring it on the surface of the mold prepared in step (1), and baking at 65°C overnight; After cooling, slowly tear off the PDMS on the template and set it aside.

Example 3 Construction of a bionic model simulating the microenvironment of lung cancer cell metastasis in vivo

1. 2D culture of lung cancer cells in a microfluidic chip

(1) The microfluidic chip is sterilized by ultraviolet irradiation, and the porous PDMS membrane is coated with BME. The specific coating process is: pretreatment of the chip, so that cells can better adhere to the porous membrane surface of the chip. Dilute BME (Cultrexbasement membrane extract, R&D Systems, McKinley Place, MN, USA) at a ratio of 1:10, mix well, inject into the sample inlet of the microfluidic chip with a micro-sampler, and wait overnight in the incubator for the gel to solidify.

(2) The microfluidic chip is turned over, that is, the third PDMS substrate faces upward. Collect the suspended mononuclear cells into a centrifuge tube, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and add fresh medium to prepare a cell suspension. Inject the mononuclear cell suspension into the channel 21 through the inlet 22, so that it is planted on the porous membrane PDMS membrane at a density of 10 ³ /cm ₂ , and then use a syringe pump to inject the PMA medium at a volumetric flow rate of 24 mm/h.

(100ng/ml) is injected into the microfluidic chip through the inlet 22, and excess medium is discharged through the outlet 23. Tilt the microfluidic chip to allow the cells to move to the side of the channel 21 . Tilt the chip 30 degrees to one side so that monocytes settle to one side of the central culture channel, and culture in a 37°C, 5% CO ₂ incubator. After 48h, monocytes were stimulated into macrophages.

(3) After the monocytes were stimulated to become M0 macrophages, the PMA medium was replaced with a normal medium (1640 medium).

(4) Plant human lung fibroblasts at a density of 10 ⁴ /cm ₂ to the position where M0 macrophages grow (when the cells proliferate to the logarithmic growth phase, digest with 0.25% trypsin, add fresh medium Blow into a cell suspension, centrifuge at 1000rpm for 5 minutes, discard the supernatant, add fresh medium to make a fibroblast suspension. Inoculate lung fibroblasts WI38 into macrophages at a cell density of 10 ⁴ cells/cm ₂ The ipsilateral side of the cells was attached to the surface of the porous membrane, and placed in a 37°C, 5% CO ₂ incubator for 4 hours under static conditions.

(5) Return the microfluidic chip to a horizontal state, inject the HUVEC suspension of vascular endothelial cells into the channel 21 through the inlet 22, and plant it on the porous PDMS membrane at a density of 10 ⁴ cells/cm ₂ , and adhere to the The other side of channel 21.

(6) After the cells on the lower side of the porous membrane of the chip are all attached to the wall, flip the chip from the upper side through the inlet 2 to inject bronchial epithelial cells 16HBE into the chip at a cell density of 10 ⁴ cells/ _cm2 , and statically make it adhere to the membrane surface After 4 hours, gently suck the culture medium from the upper channel through the outlet 2, continue to pump the mixed culture medium through the inlet continuously, and place it in a 37°C, 5% _CO2 incubator for culture. The medium is removed through outlet 13.

(7) After the above-mentioned cells are overgrown, the lung cancer cell suspension is injected into the channel 11 through the inlet 12, so that it is planted on the porous PDMS membrane at a density of 10 ³ cells/cm ₂ and inoculated in the bronchial epithelial cell area on the upper layer of the chip, Let it stand for 4 hours to make it stick. The medium is removed through outlet 13.

2. 3D culture of brain tissue cells, bone tissue cells, and liver tissue cells in microfluidic chips

(1) Digest astrocytes, osteoblasts and hepatocytes (purchased from the Cell Center of Shanghai Academy of Biological Sciences, Chinese Academy of Sciences) with trypsin and resuspend them in ice-cold cell basement membrane extract mixture (R&D, Supplementary Cat. No.), respectively mix the cell suspension with equal volumes of BME.

(2) The microfluidic chip was placed in a 37°C environment for 30 minutes to gel the BME. The culture medium is injected into the microfluidic chip through the inlet 22 and pumped continuously. At the same time, the outlet 23 is sealed to allow excess culture medium to be discharged from the inlets 214 , 215 , 216 .

(4) The microfluidic chip was placed in a 37°C incubator with a humidity of 95% and a carbon dioxide concentration of 5% for cultivation (the vacuum pump parameter was physiological cyclic strain (10% at 0.2Hz). The incubation time was 24h, and subsequent experiments were carried out.

Example 4 Detection of the effectiveness of the bionic model for simulating the metastatic microenvironment of lung cancer cells in vivo

Evaluate the effectiveness of the bionic model of the simulation body lung cancer cell transfer microenvironment constructed in Example 3, and complete it through the following experiments:

1. Detection of cell viability

Detection method: absorb the culture medium in the channel in the chip system, inject PBS into the chip channel, and wash the cells of different treatment groups twice; then pump H33342 (1:100) into the staining for 15 minutes, and wash twice with PBS solution; Pump in PI staining (1:200) for 5 minutes, wash with PBS solution twice; under a microscope, observe the fluorescence intensity under the excitation of the corresponding excitation light and record it by taking pictures.

2. Detection of co-localization of lung cancer cells and bronchial epithelial cells

The cell tracer C7000CM-DiL marked the lung cancer cells in red: the lung cancer cells were resuspended in the C7000CM-DiL working solution (1 μg/μL) before chip inoculation and incubated at 37°C for 5min, then placed at 4°C for 15min.

3. Observation of the growth pattern of human bronchial epithelial cells and vascular endothelial cells

Immunostaining for E-cadherin was used to examine the tightness of junctions between bronchial epithelial cells and vascular endothelial cells in a microfluidic chip. E-cadherin immunostaining procedure is as follows:

a. Use PBS to wash the bronchial epithelial cells and vascular endothelial cells on both sides of the media of the microfluidic chip;

b. Fix the cells with 4% paraformaldehyde for 15 minutes, permeate the membrane with 0.5% PBST solution for 10 minutes, and wash with PBS solution twice;

c. Inject the diluted blocked serum into the culture pool of the chip, put it in a wet box, and incubate at 37°C for 1 hour; dilute the stock solution of the blocked serum with freshly prepared 5% BSA, dilute it at a ratio of 1:100, and mix it with a shaker. uniform;

d. Incubate for 2 h with E-cadherin antibody (Abcam) dilution solution with a final concentration of 2 μg/mL;

e. Incubate for 1 h with Alexa 594-coupled secondary antibody;

f. Stain with DAPI at a final concentration of 10 μg/mL for 15 minutes; use a fluorescent microscope to take pictures.

4. Detect the characteristics of lung cancer cells, cancer-related fibroblasts, and macrophages

Immunostaining was used to detect the expression of tumor cell marker protein CEA, the expression of fibroblast marker protein α-SMA, and the expression of macrophage marker protein CD206, and the labeling results were presented by immunofluorescence microscopy.

The immunostaining procedure was as follows:

a. Use PBS to wash the lung cancer cells, fibroblasts and macrophages in the microfluidic chip;

b. Fix the cells with 4% paraformaldehyde for 15 minutes, permeate the membrane with 0.5% PBST solution for 10 minutes, and wash with PBS solution twice;

c. Inject the blocking serum dilution into the chip culture pool, put it in a wet box, and incubate at 37°C for 1 hour. Dilute the goat blocking serum stock solution with freshly prepared 5% BSA, dilute it at a ratio of 1:100, and mix it with a shaker;

e. Incubate for 2 h with antibodies against human CEA protein (1:100, abcam), antibodies against human α-SMA protein (1:200, Santa Cruz), and antibodies against human CD206 protein (1:50, abcam);

f. Incubate with Alexa 488 or 594-coupled secondary antibody for 1 hour; use DAPI at a final concentration of 10 μg/mL for 15 minutes; use a fluorescent microscope to take pictures.

2. Experimental results:

2.1 The result of cell viability: the cell viability is over 95% (Fig.8E-F).

2.2 The results of immunofluorescence cell tracker expression assay showed that lung cancer cell A549 and bronchial epithelial cell 16HBE co-localized (Fig. 9C-D).

2.3 E-cadherin protein immunostaining, the experimental results showed that bronchial epithelial cells 16HBE and vascular endothelial cells HUVEC were closely connected with each other (Fig.8G-H).

2.4 The expression of CEA in lung cancer cells (Fig.9E-F); the expression of α-SMA in fibroblasts and the expression of CD206 in macrophages prove that the interaction between lung cancer cells and stromal cells leads to the activation of stromal cells, and fibroblasts Transformed into cancer-associated fibroblasts α-SMA, macrophages transformed into cancer-associated macrophages expressing CD206 (Fig. 9G, 9H, 9I, 9J).

Example 5 Using a bionic model that simulates the microenvironment of lung cancer cell metastasis in vivo to monitor the process of lung cancer cell metastasis

1. Steps

1.1 Detection of epithelial-mesenchymal transition (EMT) in lung cancer cells

By detecting the expression of epithelial-mesenchymal transition (EMT) marker proteins: E-cadherin, N-cadherin, Snail1, Snail2, to evaluate whether the lung cancer cells have undergone epithelial-mesenchymal transition.

The immune labeling process is as follows:

a, using PBS to wash the lung cancer cells on the porous membrane;

b. Fix the cells with 4% paraformaldehyde for 15 minutes, permeate the membrane with 0.5% PBST solution for 10 minutes, and wash with PBS solution twice;

c. Inject the diluted blocked serum into the culture pool of the chip, put it in a wet box, and incubate at 37°C for 1 hour; dilute the stock solution of the blocked serum with freshly prepared 5% BSA, dilute it at a ratio of 1:100, and mix it with a shaker. uniform;

d. Use anti-human E-cadherin antibody (1:100, Proteintech), anti-human N-cadherin antibody (5 μg/ml, abcam), anti-human Snail1 protein antibody (1:50, abcam), anti-human Snail2 protein antibody (1:400, CellSignaling Technology), incubated for 2h;

f. Incubate with Alexa 488 or 594-coupled secondary antibody for 1 hour; use DAPI at a final concentration of 10 μg/mL for 15 minutes; use a fluorescent microscope to take pictures.

1.2 Identification of the growth pattern of metastatic lung cancer cells

Lung cancer cells were labeled with C34557; C34557 working solution (10 μmol/L) was added to the 1640 cell culture medium, and the cells were incubated for 20 min; then incubated with fresh medium for 5 min, repeated 4 times. (Lung cancer cells were marked green with the cell tracer C34557 in advance: resuspend the lung cancer cells in C34557 working solution (10 μmol/L) and incubate at 37°C for 20 min before chip inoculation, and then resuspend in 4 times the volume of culture medium at 37°C Incubate for 5min).

1.3 Detection of invasion of metastatic lung cancer cells

Whether metastatic lung cancer cells can invade into multiple distant organs on the microfluidic chip was evaluated by detecting the expression of tissue damage marker proteins. Immunostaining was performed with antibodies against human brain tissue damage marker protein CXCR4 (1:100, abcam), anti-human bone tissue damage marker protein RANKL antibody (1:50, Santa Cruz Biotechnology), and anti-human liver tissue damage marker protein AFP antibodies.

The immunostaining procedure was as follows:

a. Wash the cells twice with PBS;

b. Fix the cells with 4% paraformaldehyde for 15 minutes, permeate the membrane with 0.5% PBST solution for 10 minutes, and wash with PBS solution twice;

c. Inject the diluted blocked serum into the culture pool of the chip, put it in a wet box, and incubate at 37°C for 1 hour; dilute the stock solution of the blocked serum with freshly prepared 5% BSA, dilute it at a ratio of 1:100, and mix it with a shaker. uniform;

e. Using the above primary antibody, incubate for 2 hours;

f. Incubate for 1 h with Alexa 488 or 594-coupled secondary antibody; stain with DAPI at a final concentration of 10 μg/mL for 15 minutes; take pictures with a fluorescent microscope.

2. Experimental results

2.1 Epithelial-mesenchymal transition of lung cancer cells

After the lung cancer cells and cancer-related mesenchymal cells were cultured for 4 days, the lung cancer cells expressed EMT, and the expression of three EMT markers (N-Ca, Snail1, Snail2) indicated that the lung cancer cells had undergone epithelial-mesenchymal transition (Fig.10A-B).

2.2 Long distance metastasis of lung cancer cells

Cell counts showed that the number of non-small cell lung cancer cells A549 adhered to the porous membrane above the brain tissue cells, bone tissue cells, and liver tissue cells were 148±8, 364±16, and 299±13; The number of lung cancer cells H446 adhered to the porous membrane above brain tissue cells, bone tissue cells, and liver tissue cells were 255±16, 128±8, and 278±18, respectively (Fig.11A). The above results indicated that the tropism of A549 cell metastasis was bone tissue>liver tissue>brain tissue; the tropism of H446 cell metastasis was liver tissue>brain tissue>bone tissue, and these features were similar to the clinical features of lung cancer patients.

2.3 Growth patterns of metastatic lung cancer cells in multiple distant organs

Cell morphology was observed with an inverted fluorescence microscope. As shown in Fig.11B, the morphology of the cells transferred to the distant target organ was clustered.

2.4 Invasion of metastatic lung cancer cells

The results are shown in Fig.12A and Fig.12B, CXCR4 is expressed in astrocyte H1800, RANKL is expressed in osteoblast Fob, and AFP is expressed in liver cell L-02, indicating that metastatic lung cancer cells have invaded target organ cells middle.

Through the above examples, it is found that the microfluidic chip of the present invention can effectively simulate the metastasis and invasion process of lung cancer cells in vivo, and provide a basis for clinical treatment plan.

Although the embodiments of the present invention have been shown and described, those skilled in the art can understand that various changes, modifications, substitutions and modifications can be made to these embodiments without departing from the principle and spirit of the present invention. The scope of the invention is defined by the claims and their equivalents.

Claims (9)

1. it is a kind of for simulate interior tumor cell and its transfer microenvironment bionical micro-fluidic chip, it is characterised in that it is described Micro-fluidic chip is closed by one of three layers of PDMS substrates and the interlaced irreversible sealing-in of the porous PDMS films of two-layer It is overall；Ground floor PDMS substrates (1) is provided with the air duct (11) for supplying air circulation, the ground floor liquid for passing in and out for liquid and enters Mouthful (12) and ground floor liquid outlet (13), respectively positioned at the air duct (11) both sides the first vacuum passage (14) it is upper The top half of half part and the second vacuum passage (15)；Second layer PDMS substrates (2) is provided with the liquid for circulating for liquid and leads to Road (21), the second layer liquid inlet (22) for passing in and out for liquid and second layer liquid outlet (23) are logical positioned at the liquid respectively The latter half of first vacuum passage (14) of road (21) both sides and the latter half of the first vacuum passage (15), the liquid lead to The side in road (21) is provided with three interface channels for stretching out, respectively the first interface channel (211), the second interface channel (212), the 3rd interface channel (213), first interface channel (211), second interface channel (212), the described 3rd The end of interface channel (213) is respectively equipped with first entrance (214), second entrance (215), the 3rd entrance (216)；Third layer PDMS substrates (3) are provided with the first cell culture chamber (31), the second cell culture chamber (32), the training of the 3rd cell for cell culture Support room (33)；First cell culture chamber (31), second cell culture chamber (32), the 3rd cell culture chamber (33) It is located under first interface channel (211), second interface channel (212), the 3rd interface channel (213) respectively Side, by the first porous PDMS films (5) and first interface channel (211), second interface channel (212), described the Three interface channels (213) are communicated；The top half of first vacuum passage (14), the upper half of second vacuum passage (15) Part is corresponding with the latter half structure of the latter half of first vacuum passage (14), second vacuum passage (15) Form first vacuum passage (14) and second vacuum passage (15) of structural integrity；The ground floor liquid inlet And the ground floor liquid outlet (13) is respectively arranged at the upstream and downstream and homonymy of the air duct (11) (12)；Described second Layer liquid inlet (22) and the second layer liquid outlet (23) be respectively arranged at the fluid passage (21) upstream and downstream and together Side, the air duct (11) positioned at the surface of the fluid passage (21), the opening of the air duct (11) with it is described The opening of fluid passage (21) is relative and is separated by the second porous PDMS films (4)；The air duct (11) is covered in described The upstream end of fluid passage (21), first interface channel (211), second interface channel (212), the 3rd connection Downstream of the passage (213) positioned at the fluid passage (21).

2. micro-fluidic chip according to claim 1, it is characterised in that the first porous PDMS films (5) and described The thickness of two porous PDMS films (4) is 10 μm, aperture is 10 μm.

3. micro-fluidic chip according to claim 1, it is characterised in that the air duct (11) and the fluid passage (21) cross section is rectangle, and size is：10mm long × 4mm wide, 35mm long × 4mm wide；First vacuum passage (14), The cross section of second vacuum passage (15) is rectangle, and size is：7mm long, width 2mm；First cell culture chamber (31), second cell culture chamber (32), the shape of cross section of the 3rd cell culture chamber (33) are rectangle, size For：1.5mm high, width 1.5mm.

4. micro-fluidic chip according to claim 1, it is characterised in that the length of the air duct (11) is 10mm； The length of the fluid passage (21) is 35mm；It is first interface channel (211), second interface channel (212), described Distance of 3rd interface channel (213) apart from the upstream end of the fluid passage (21) is 2.2mm.

5. micro-fluidic chip according to claim 1, it is characterised in that the ground floor liquid inlet (12) and described The distance of one layer of liquid outlet (13) is 15mm；The second layer liquid inlet (22) and the second layer liquid outlet (23) Distance is 15mm.

6. micro-fluidic chip according to claim 1, it is characterised in that first interface channel (211), described second Interface channel (212), the 3rd interface channel (213) are parallel to each other.

7. micro-fluidic chip according to claim 1, it is characterised in that first vacuum passage (14) and the air The distance of passage (11) is 2mm；Second vacuum passage (15) is 2mm with the distance of the air duct (11).

8. a kind of preparation method of the micro-fluidic chip any one of claim 1-7, it is characterised in that the preparation side Method is comprised the following steps：

(1) the SU-8 sun with the microchannel in the micro-fluidic chip any one of claim 1-7 and micro-structural is prepared Mould；

(2) the SU-8 formpistons for being prepared with step (1), as template, are raw material duplication with PDMS, are prepared into any in claim 1-7 Ground floor PDMS substrates, second layer PDMS substrates, third layer PDMS substrates described in；

(3) the porous PDMS films described in two claims 1 are prepared；

(4) the ground floor PDMS substrates, the second layer PDMS substrates, the third layer PDMS bases for preparing step (2) Two porous interlaced placement sealing-ins of PDMS films prepared by piece and step (3) are formed.

9. preparation method according to claim 8, it is characterised in that the preparation method is comprised the following steps：

(1) it is micro- logical in the micro-fluidic chip any one of use computer aided design software CAD drafting claims 1-7 Road and micro-structural；Drafting figure is printed upon on SU-8 films as mask, mould is made using standard photolithography process；

(2) by PDMS and curing agent in mass ratio 10：1 is mixed, and the die surface for being coated in step (1) preparation is poured after vacuumizing, 80 DEG C of baking 1h；

(3) slowly PDMS is torn in template after cooling down, entrance, outlet is got out in PDMS substrates relevant position, then cut Into suitable size；

(4) porous PDMS film productions are PDMS and curing agent in mass ratio 15：1 mixes, sol evenning machine 3000rpm, 1 minute, takes out true The die surface for being coated in step (1) preparation is poured after sky, 65 DEG C are baked overnight；Slowly PDMS is torn in template after cooling, It is standby.