Construction and application of tumor microenvironment chip

Abstract

The invention discloses construction and application of a tumor microenvironment chip, and belongs to the technical field of micro-fluidic chips. According to the tumor microenvironment chip, a micro-fluidic chip mold is prepared through a 3D printing technology, then mold reversing, demolding and sealing are conducted on the chip mold through PDMS, a PDMS micro-fluidic chip is obtained, then A549 cells and HUVECs are co-cultured on the PDMS micro-fluidic chip, a vascularized tumor chip is prepared, drug resistance of the vascularized tumor chip is induced through platelets, and the tumor microenvironment chip is obtained. And preparing the tumor microenvironment chip. The tumor microenvironment chip designed by the invention can simulate a continuous and naturally flowing drug delivery condition in an in-vivo blood supply environment, and is convenient for simulating a bionic tumor microenvironment to carry out experiments, real-time observation and drug screening.

Description

Technical Field

[0001] The present invention belongs to the technical field of microfluidic chips, and particularly relates to the construction and application of a tumor microenvironment chip. Background Art

[0002] Currently, there are many limitations in existing preclinical drug screening models. For example, for animal models that are very important for in vivo preclinical evaluation, the commonly used subcutaneous implantation model lacks the characteristics of a natural tissue-specific microenvironment. And problems such as interspecies differences greatly limit the evaluation of drug efficacy and toxicity, resulting in poor predictive ability for treatment responses in human clinical trials. In addition, animal models also have problems such as ethics, economy, and reproducibility. In vitro cell culture models are used to solve the problems existing in animal models. Traditional 2D cell culture models have the characteristics of low cost and high throughput, but their predictive value for drug responses is limited because they cannot generalize the in vivo tumor microenvironment (TME), which may amplify the sensitivity of tumor cells to drugs. 3D spheroid and organoid models have obvious improvements based on 2D cell culture models, but there are still various problems. For example, statically cultured spheroids / organoids cannot reproduce shear stress / hydration pressure and tissue interactions through blood / interstitial flow and vascular perfusion, nor can they reconstruct the inherent functions of in situ organs (such as intestinal peristalsis, blood flow, etc.), which may affect drug delivery behavior. The evaluation of the efficacy of anti-drug-resistant oxaliplatin administration usually uses cytotoxicity experiments on drug-resistant cell lines. However, drug-resistant cell lines are difficult to culture, require continuous drug induction, and their drug-resistant properties are unstable, bringing great difficulties to in vitro evaluation.

[0003] Microfluidic chip technology is one of the important scientific and technological frontiers in the 21st century, providing an important platform for evaluating the in vivo delivery behavior of drugs in complex tumor models. Tumor microenvironment chips are a typical type of microfluidic chip. To address the many deficiencies in simple in vitro models and animal experiments, the concept of tumor microenvironment chips was proposed.

[0004] In addition to the characteristics of miniaturization, integration, and low power consumption, tumor microenvironment chip technology can also precisely control multiple system parameters. Such as fluid shear stress, and construct co-cultures of multiple cells, etc., to simulate the complex structure, microenvironment, and physiological functions of human tumor sites. Therefore, study coffee. Summary of the Invention

[0005] To solve the problems existing in the background technology, the purpose of the present invention is to provide a construction and application of a tumor microenvironment chip. On the one hand, the present invention solves the problem that the existing microfluidic chips cannot simulate the continuous natural flow drug delivery conditions in the in vivo blood supply environment when applied to cell culture, and lack the ability to regulate the tumor microenvironment in real time and continuously to observe the drug effects. On the other hand, the present invention can effectively reduce the problems such as long cycle and poor stability brought by animal experiments.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] In the first aspect, the present invention provides a tumor microenvironment chip, which is obtained by co-culturing vascular endothelial cells in the microchannels of the upper chip of a PDMS microfluidic chip, co-culturing tumor cells in the microchannels of the lower chip, and inducing epithelial-mesenchymal transition (EMT) of tumor cells with platelets and creating a drug-resistant environment; the PDMS microfluidic chip includes an upper chip, a porous membrane and a lower chip from top to bottom in sequence. The upper chip and the lower chip are closely attached. Parallel microchannels are provided on the upper chip, and parallel microchannels are provided on the lower chip. The microchannels are closely connected. The microchannels of the upper chip and the microchannels of the lower chip are completely coincident in the vertical direction. Liquid inlets and liquid outlets are respectively provided at both ends of each microchannel of the upper chip. The liquid inlets are communicated with the entrances of the microchannels, and the liquid outlets are communicated with the exits of the microchannels. Liquid inlets and liquid outlets are respectively provided at both ends of each microchannel of the lower chip. The liquid inlets are communicated with the entrances of the microchannels, and the liquid outlets are communicated with the exits of the microchannels.

[0008] Based on the above technical solution, further, the length of the microchannel is 10 - 20 mm, the width is 1 - 5 mm, and the height is 0.1 - 1 mm.

[0009] Based on the above technical solution, further, 3 - 50 parallel microchannels are provided on the upper chip and the lower chip.

[0010] In the second aspect, the present invention provides a construction method of the above tumor microenvironment chip, including the following steps:

[0011] 1) Use a stereolithography 3D printer to manufacture an upper chip mold and a lower chip mold;

[0012] 2) Pour the mixed solution of PDMS prepolymer and curing agent into the upper chip mold and the lower chip mold respectively, and then use a vacuum dryer to degas for 0.5 - 2 hours;

[0013] 3) Place the PDMS material in an oven for curing. Take out the upper chip template and the lower chip template. Fix the transparent polyester film on each microchannel of the lower chip. After both the upper chip and the lower chip have been treated with corona plasma, closely attach them vertically and bake them together to obtain the PDMS microfluidic chip;

[0014] 4) Prepare a cell suspension of vascular endothelial cells and inject it into the microchannels of the upper chip of the PDMS microfluidic chip through the liquid inlet, and incubate it at 36 - 38 °C for 10 - 48 hours;

[0015] 5) Prepare a cell suspension of tumor cells and inject it into the microchannels of the lower chip of the PDMS microfluidic chip through the liquid inlet. Flip the chip and incubate it at 36 - 38 °C for 10 - 48 hours;

[0016] 6) Disperse platelets in the culture medium and continuously inject it into the microchannels of the upper chip of the PDMS microfluidic chip through the liquid inlet, and continuously culture it dynamically for 60 - 100 h to obtain the product.

[0017] Based on the above technical solution, further, in step 3), the curing temperature is 60 - 70 °C, the curing time is 20 - 30 hours; the baking temperature is 75 - 85 °C.

[0018] Based on the above technical solution, further, the vascular endothelial cells in step 4) are HUVECs cells.

[0019] Based on the above technical solution, further, in steps 4) and 5), the concentration of the cell suspension is 0.1 - 10×10 6 / mL, and the injection volume of the cell suspension is 10 - 50 μL.

[0020] Based on the above technical solution, further, the tumor cells in step 5) include lung cancer, melanoma, thyroid cancer, pancreatic cancer, skin cancer, gallbladder cancer, nasopharyngeal carcinoma, gastric cancer, esophageal cancer, breast cancer, ovarian cancer, kidney cancer, bladder cancer, cervical cancer, prostate cancer, rectal cancer, and colorectal cancer.

[0021] Based on the above technical solution, further, the tumor cells in step 5) are A549 cells.

[0022] Based on the above technical solution, further, in step 6), the concentration of platelets is 1 - 10×10 5 / mL, and the flow rate is controlled at 5 - 50 μL / min.

[0023] In the third aspect, the present invention provides the application of the above tumor microenvironment chip in evaluating the administration effectiveness of chemotherapeutic drugs in vitro.

[0024] Based on the above technical solutions, further, the chemotherapeutic drug includes oxaliplatin.

[0025] The beneficial effects of the present invention compared with the prior art are as follows:

[0026] 1. The tumor microenvironment chip of the present invention is prepared by using a PDMS microfluidic chip, and the PDMS microfluidic chip has good biocompatible performance; in addition, the tumor microenvironment chip is simple to construct, has strong reproducibility, can keep cells in good vitality, and simulate the tumor microenvironment.

[0027] 2. The tumor microenvironment chip of the present invention can simulate an evaluation model of tumor drug resistance environment characteristics, realize the accurate evaluation and selection of chemotherapeutic drugs represented by oxaliplatin, can construct a complex pathophysiological environment in the patient body, and provides a very promising method for the evaluation and screening of chemotherapeutic drugs. It can display the interaction between tumor cells and related cells in the 3D TME, thereby reflecting the dynamic process involved in tumor development. In addition, the tumor organ chip can reconstruct the in vivo system in a simple and inexpensive way, so as to achieve high-throughput multiple drug screening at the organ and whole body levels, and the response of the treatment effect is more accurate and sensitive, so it is beneficial to the development of the chemotherapeutic drug delivery system; platelets can release many bioactive substances into the blood, and then enhance the interaction between platelets and cancer, stimulate cancer metastasis and chemotherapy resistance. Design a vascularized tumor organ chip that promotes drug resistance by platelets, simulate the in vivo tumor environment, create a drug resistance environment, and perform dynamic drug administration. Combine dynamic organ chips and in vivo animal models to conduct in vitro evaluation at multiple levels and multiple models. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention, the drawings related to the embodiments will be briefly introduced below.

[0029] Figure 1 It is a schematic cross-sectional view of the PDMS microfluidic chip of Embodiment 1 of the present invention.

[0030] Figure 2 It is a schematic diagram of the tumor microenvironment chip of Embodiment 2 of the present invention.

[0031] Figure 3 It is a result diagram of the expression levels of TGF-β content (A), E-cadherin (B), and MMP (C) in the tumor microenvironment chip of Embodiment 3 of the present invention.

[0032] Figure 4 It is a diagram of the morphology and proliferation of tumor cells in the tumor microenvironment chip (TMEOC) of Embodiment 4 of the present invention under bright field of a confocal microscope (A) and a result diagram of TMEOC for evaluating the efficacy of oxaliplatin (B). DETAILED DESCRIPTION OF THE EMBODIMENTS

[0033] The present invention will be described in detail below in conjunction with embodiments. However, the implementation manners of the present invention are not limited thereto. Obviously, the embodiments described below are only partial embodiments of the present invention. For those skilled in the art, without creative efforts, obtaining other similar embodiments will fall within the protection scope of the present invention.

[0034] Embodiment 1: Preparation of PDMS Microfluidic Chip

[0035] The schematic diagram of the PDMS microfluidic chip is as Figure 1 shown, which successively includes an upper chip, a porous membrane, and a lower chip from top to bottom. The upper chip and the lower chip are closely attached. There are 10 parallel microchannels arranged on the upper chip, and 10 parallel microchannels arranged on the lower chip. The microchannels are closely connected. The microchannels are 15 mm in length, 1.5 mm in width, and 0.5 mm in height. The microchannels on the upper chip and the microchannels on the lower chip are completely coincident in the vertical direction. At both ends of each microchannel on the upper chip, a liquid inlet and a liquid outlet are respectively arranged. The liquid inlet is communicated with the entrance of the microchannel, and the liquid outlet is communicated with the exit of the microchannel. At both ends of each microchannel on the lower chip, a liquid inlet and a liquid outlet are respectively arranged. The liquid inlet is communicated with the entrance of the microchannel, and the liquid outlet is communicated with the exit of the microchannel.

[0036] The preparation process is as follows:

[0037] 1) Use a stereolithography 3D printer to manufacture the upper chip mold and the lower chip mold;

[0038] 2) Mix the PDMS prepolymer and the curing agent, and pour them into the above-mentioned upper chip mold and lower chip mold respectively. Then use a vacuum dryer to degas for 1 hour;

[0039] 3) Cure the PDMS material in an oven at 65 °C for 24 hours. After curing, take out the upper chip template and the lower chip template, trim and clean them; fix the transparent polyester (PET) film on each microchannel of the lower chip. Both the upper chip and the lower chip are vertically and closely attached after being treated by corona plasma, and are bonded together by baking in an oven at 80 °C to obtain the PDMS microfluidic chip.

[0040] Embodiment 2: Preparation of Tumor Microenvironment Chip

[0041] The preparation of the tumor microenvironment chip includes the following steps:

[0042] (1) Prepare a cell suspension of HUVECs cells with a density of (1×10 6 / ml), inject 20 ul of the cell suspension into the upper channel of the chip, and incubate at 37 °C for 24 h.

[0043] (2) Configure A549 cells into a cell suspension with a density of 1×10 6 / mL. Take 20 μL of the cell suspension and inject it into the lower channel of the chip. Flip the chip and continue to incubate at 37 °C for 24 h.

[0044] (3) Collect mouse platelets and disperse them in the culture medium at a concentration of 200*10 3 / mL. Perfuse the platelet-containing culture medium into the upper channel at a flow rate of 10 μL / min for continuous dynamic culture for 72 h. Use platelets to induce and simulate the tumor epithelial-mesenchymal transition (EMT) process and create a drug-resistant environment to prepare a drug-resistant tumor microenvironment chip.

[0045] A schematic diagram of the tumor microenvironment chip is as shown in Figure 2 the figure.

[0046] Example 3: Functional characterization of the tumor microenvironment chip

[0047] After the successful establishment of the tumor microenvironment chip, using the vascular tumor chip (VTOC) without perfusion of platelet-containing culture medium as a control, use a confocal microscope in bright field to observe the morphology of A549 lung cancer cells in the lower tumor channel of the tumor microenvironment chip (TMEOC), and investigate the morphological transformation of cells between epithelial and mesenchymal states.

[0048] Verification of proliferation ability. The specific experimental process is as follows: After the successful establishment of the chip, perfuse PBS and the culture medium containing QXA sol (2 μM) into the upper vascular channels of the vascular tumor chip (VTOC) and the vascular tumor microenvironment chip (TMEOC) induced by adding platelets at a flow rate of 10 μL / min for dynamic culture for 72 h. Remove the chip from the microfluidic device, wash the lower channel 3 times with cold PBS, inject 4% paraformaldehyde into the lower channel, incubate in the dark at room temperature for 20 min for fixation, and then wash the channel 3 times with cold PBS. Dilute the Ki67 primary antibody 100 times with PBS containing 1% BSA and 0.1% Triton-X and inject it into the lower channel of the chip, and incubate overnight at 4 °C. After incubation, wash the channel 3 times again with cold PBS, inject the secondary antibody diluted 100 times with PBS containing 1% BSA and 0.1% Triton-X, incubate in the dark at room temperature for 2 h, and then wash the channel 3 times with cold PBS. Inject Hoechst 33342 staining solution into the lower channel to incubate for 10 min to stain the nuclei, and finally wash the channel 3 times with cold PBS and inject 20 μL of anti-fluorescence quenching agent. Use a laser confocal microscope to observe the fluorescence intensity of the cell nuclei.

[0049] After the addition of platelets, the tumor cells showed a heterogeneous phenotype, and some of the cells became elongated and the proliferation rate increased significantly.

[0050] Increased cancer cell proliferation caused by phenotypic changes through EMT leads to chemoresistance, which is affected by platelets. TGF-β released by platelets is crucial for EMT of tumor cells. During the EMT transformation of tumors, the expression of E-cadherin is often reduced, and the expression of MMP protein increases. E-cadherin and MMP are important markers of the mesenchymal and epithelial states of tumor cells. Here, the expression of E-cadherin and MMP-9 proteins in tumor cells was detected. The specific experimental process is as follows: The tumor microenvironment chip TMEOC after culture and the vascular tumor chip VTOC not perfused with platelet-containing medium were removed from the microfluidic device, and the upper channels were washed 3 times with cold PBS. 4% paraformaldehyde was injected into the upper channels, and after incubation in the dark at room temperature for 20 min for fixation, the channels were washed 3 times with cold PBS. The E-cadherin primary antibody was diluted 100 times with PBS containing 1% BSA and 0.1% Triton-X and then injected into the upper channels of the chip, and incubated overnight at 4°C. After incubation, the upper channels were washed 3 times again with cold PBS. The secondary antibody diluted 100 times with PBS containing 1% BSA and 0.1% Triton-X was injected into the upper channels, and after incubation in the dark at room temperature for 2 h, the upper channels were washed 3 times with cold PBS. Hoechst33342 staining solution was injected into the upper channels and incubated for 10 min to stain the nuclei. Finally, the upper channels were washed 3 times with cold PBS, and 20 μL of anti-fluorescence quencher was injected. Observation was performed using a laser confocal microscope. The tumor cell culture medium in each group of chips was collected, and the content of MMP-9 in it was quantified using an MMP-9 enzyme-linked immunosorbent assay kit. The experiment used recombinant human MMP-quantitative sandwich enzyme immunoassay technology to generate antibodies against the recombinant protein, and the optical density was read using a double-wavelength 450 nm microplate reader to achieve the purpose of quantification and determine the content of MMP-9.

[0051] The results are as Figure 3 shown. The concentration of TGF-β in the platelet-induced tumor microenvironment chip (TMEOC) increased in a time-dependent manner relative to VTOC. In addition, after 72 h of platelet induction, the expression level of E-cadherin in tumor cells in TMEOC decreased, and the expression level of MMP-9 increased.

[0052] Example 4: Evaluation of the anti-tumor activity of oxaliplatin using TMEOC

[0053] Given the role of platelets in the tumor microenvironment, freshly isolated platelets (about 2×10 5Platelets (1×10⁶ cells / μL) were injected into the vascular channels of VTOC for three days to establish the Tumor Microenvironment-on-a-Chip (TMEOC). After platelet injection, a time-dependent increase in TGF-β concentration in the platelet-induced TMEOC was observed relative to VTOC. In addition, 72 h after platelet induction, the expression level of E-cadherin in tumor cells in TMEOC was downregulated, and the expression level of MMP-9 was upregulated. After treatment, the tumor cells presented a heterogeneous phenotype, and some of the cells became elongated with a significantly increased proliferation rate.

[0054] The results were as Figure 4 shown. After administration of platelets, the tumor cells presented a heterogeneous phenotype, and some of the cells became elongated with a significantly increased proliferation rate (4A). When treated with the anticancer drug oxaliplatin (2 μM / mL), significant proliferation inhibition and anticancer effects were observed in VTOC, while in TMEOC, the therapeutic effect was limited and drug-resistant cells were present ( Figure 4 B). These results indicate that the TMEOC invented by us can effectively mimic tumor EMT and create a drug-resistant environment.

[0055] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A tumor microenvironment chip, characterized in that: The tumor microenvironment chip is obtained by co-culturing vascular endothelial cells in the microchannel of the upper chip of the PDMS microfluidic chip, co-culturing tumor cells in the microchannel of the lower chip, inducing epithelial-mesenchymal transition (EMT) of tumor cells with platelets and creating a drug-resistant environment; the PDMS microfluidic chip comprises an upper chip, a porous membrane and a lower chip from top to bottom, the upper chip and the lower chip are tightly fitted, the upper chip is provided with parallel microchannels, the lower chip is provided with parallel microchannels, the microchannels are closely connected, the microchannels of the upper chip and the microchannels of the lower chip are completely overlapped in the vertical direction, each microchannel of the upper chip is provided with a liquid inlet and a liquid outlet at both ends, the liquid inlet is connected to the inlet of the microchannel, and the liquid outlet is connected to the outlet of the microchannel, and each microchannel of the lower chip is provided with a liquid inlet and a liquid outlet at both ends, the liquid inlet is connected to the inlet of the microchannel, and the liquid outlet is connected to the outlet of the microchannel.

2. The tumor microenvironment chip according to claim 1, characterized in that: The length of the microchannel is 10 to 20 mm, the width is 1 to 5 mm, and the height is 0.1 to 1 mm.

3. The tumor microenvironment chip according to claim 1, characterized in that: 3 to 50 parallel microchannels are arranged on the upper chip and the lower chip.

4. The method for constructing a tumor microenvironment chip according to any one of claims 1 to 3, characterized in that: The steps include: 1) Using a stereolithography 3D printer to manufacture an upper chip mold and a lower chip mold; 2) Pour the mixed solution of PDMS prepolymer and curing agent into the upper chip mold and the lower chip mold respectively, and then use a vacuum dryer to degas for 0.5 to 2 hours; 3) Put the PDMS material into an oven for curing, take out the upper chip template and the lower chip template, fix the transparent polyester film on each microchannel of the lower chip, vertically and closely fit the upper chip and the lower chip after corona plasma treatment, bake and bond them together, and obtain a PDMS microfluidic chip; 4) preparing a cell suspension of vascular endothelial cells, injecting it into the microchannel of the upper chip of the PDMS microfluidic chip through the liquid inlet, and incubating it at 36-38° C. for 10-48 hours; 5) Prepare a cell suspension of tumor cells, inject it into the microchannel of the lower chip of the PDMS microfluidic chip through the liquid inlet, flip the chip, and incubate it at 36-38° C. for 10-48 hours; 6) The platelets are dispersed in the culture medium, and are continuously injected into the microchannel of the upper chip of the PDMS microfluidic chip through the liquid inlet, and the dynamic culture is continuously performed for 60 to 100 hours.

5. The construction method according to claim 4, characterized in that: In step 3), the curing temperature is 60-70°C, the curing time is 20-30 hours, and the baking temperature is 75-85°C.

6. The construction method according to claim 4, characterized in that: The vascular endothelial cells described in step 4) are HUVECs cells.

7. The construction method according to claim 4, characterized in that: The concentration of the cell suspension in step 4) and step 5) is 0.1 to 10×10 6 / mL, and the injection volume of cell suspension is 10-50μL.

8. The construction method according to claim 4, characterized in that: The tumor cells described in step 5) include lung cancer, melanoma, thyroid cancer, pancreatic cancer, skin cancer, gallbladder cancer, rhinitis cancer, gastric cancer, esophageal cancer, breast cancer, ovarian cancer, kidney cancer, bladder cancer, cervical cancer, prostate cancer, rectal cancer and colorectal cancer.

9. The construction method according to claim 4, characterized in that: In step 6), the platelet concentration is 1 to 10×10 5 / mL, and the flow rate was controlled at 5-50μL / min.

10. Use of the tumor microenvironment chip according to any one of claims 1 to 3 in evaluating the effectiveness of chemotherapy drugs in vitro.

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