A microfluidic chip and its construction method and application

Abstract

The present invention discloses a microfluidic chip and its construction method and application. A microfluidic chip of the present invention includes a polydimethylsiloxane layer and a base layer, the polydimethylsiloxane layer contains three or more microchannels; the microchannels are connected by a plurality of parallel microgrooves; the microchannels are provided with channels connected to the outside world; the base layer is at least one of a glass base, a polydimethylsiloxane base or a polystyrene base. The preparation method of the microfluidic chip of the present invention is simple and can achieve co-culture of tumor nerve cells. The pancreatic cancer-neural-stromal cell microenvironment model constructed using the microfluidic chip of the present invention can achieve the co-construction of the pancreatic cancer stromal microenvironment and the neural microenvironment, and can also perform qualitative and quantitative evaluation of nanomedicines targeting the neural microenvironment and the stromal microenvironment, which plays an important role in the screening of drugs related to the treatment of pancreatic cancer.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a microfluidic chip and a construction method and application thereof. Background Art

[0002] In recent years, research on the regulation of the cancer microenvironment (primarily the stromal microenvironment and the neural microenvironment) has received increasing attention. This research mainly includes cells such as stellate cells, neurons, immune cells, vascular cells, and tumor cells, as well as the insoluble extracellular matrix and soluble cytokines they secrete. The neural microenvironment of pancreatic cancer influences its pathological process, mainly manifested in the ability of cancer cells and neurons to promote growth through neurotrophic factors, nerve growth factor (NGF), cytokines, and neurotransmitters, thereby triggering the occurrence of pancreatic cancer. Secondly, the connective tissue (stromal) microenvironment can regulate the occurrence and development of pancreatic cancer. When stellate cells in the stromal microenvironment are in an activated state, the dense collagen fiber tissue they secrete can hinder the arrival of drugs or immune cells. Research on the pancreatic cancer microenvironment is expected to develop nerve-related nano-drug carriers with anti-tumor properties, which is of great significance for the treatment of pancreatic cancer.

[0003] In the relevant art, pancreatic cancer treatment strategies primarily involve inhibiting neural factors and connective tissue factors. Blockers encapsulated in transferrin can inhibit the directional migration of cancer cells along axons. Nanocarriers delivering siRNA targeting NGF can suppress the expression of genes related to pancreatic cancer in orthotopic nude mice. Transforming growth factor β (TGF-β) regulates the activity of stellate cells in connective tissue, so eliminating or inhibiting TGF-β expression and secretion is another approach to inhibiting pancreatic cancer progression. Recently developed siRNA gene knockdown techniques and the CRISPR / Cas9 gene editing system enable gene editing in stellate cells. However, these technologies still have certain limitations. Naked siRNA is highly degraded in the body, making long-term therapeutic efficacy unsatisfactory. The adenoviral vectors used in CRISPR / Cas9 technology pose certain safety risks, and non-viral vectors are inefficient. Current research on the signaling pathways underlying tumor-neuron-stellate cell interactions and tumor behavior also has certain limitations. Traditional animal experimental methods for screening different drug combinations are inefficient, time-consuming, and raise ethical concerns.

[0004] Based on this, the present invention proposes a microfluidic chip that can realize the co-culture of pancreatic tumor-neuron cells and drug performance screening. It is hoped that the development of nanocarrier-loaded siRNA and CRISPR / Cas9 system can be combined with the use of microfluidic chips and automated analysis and imaging systems to improve the speed and efficiency of screening different drug combinations and different types of cancer cell systems, thereby realizing in vivo anti-tumor effect evaluation. Summary of the Invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention provides a microfluidic chip with a simple structure that can realize co-culture of pancreatic cancer-neural-stromal cells and drug performance screening.

[0006] The present invention also provides a method for constructing the microfluidic chip.

[0007] The present invention also proposes an application of the microfluidic chip in constructing a pancreatic cancer-nerve-stromal cell microenvironment model.

[0008] The present invention also proposes an application of the microfluidic chip in drug screening.

[0009] The present invention also proposes an application of the pancreatic cancer-nerve-stromal cell microenvironment model in drug screening.

[0010] A first aspect of the present invention provides a microfluidic chip, comprising a polydimethylsiloxane (PDMS) layer and a substrate layer;

[0011] The polydimethylsiloxane layer comprises two or more microchannels;

[0012] The microchannels are connected by a plurality of parallel arranged microchannels;

[0013] The base layer is at least one of a glass base layer, a polydimethylsiloxane base layer or a polystyrene base layer.

[0014] According to some embodiments of the present invention, the polydimethylsiloxane layer comprises three microchannels.

[0015] According to some embodiments of the present invention, the microchannels contained in the polydimethylsiloxane layer are arranged in parallel.

[0016] According to some embodiments of the present invention, the microchannel is provided with a channel connected to the outside world, and the channel is used for injecting cells.

[0017] According to some embodiments of the present invention, the microchannels are connected by 3-8 parallel arranged microchannels.

[0018] According to some embodiments of the present invention, the microchannels are connected by five parallel arranged microchannels.

[0019] According to some embodiments of the present invention, the substrate layer is a glass substrate layer.

[0020] According to some embodiments of the present invention, the glass substrate layer includes a bottom of a glass slide or a bottom of a glass culture dish.

[0021] According to some embodiments of the present invention, the length of the microchannel is 800-1200 microns.

[0022] According to some embodiments of the present invention, the length of the microchannel is 1000 microns.

[0023] According to some embodiments of the present invention, the width of the microchannel is 400-600 microns.

[0024] According to some embodiments of the present invention, the width of the microchannel is 500 microns.

[0025] According to some embodiments of the present invention, the height of the microchannel is 150-250 microns.

[0026] According to some embodiments of the present invention, the height of the microchannel is 200 microns.

[0027] According to some embodiments of the present invention, the length of the microchannel is 100-200 microns.

[0028] According to some embodiments of the present invention, the length of the microchannel is 150 micrometers.

[0029] According to some embodiments of the present invention, the width of the microchannel is 8-12 microns.

[0030] According to some embodiments of the present invention, the width of the microchannel is 10 micrometers.

[0031] According to some embodiments of the present invention, the height of the microchannel is 5-10 microns.

[0032] According to some embodiments of the present invention, the height of the microchannel is 7 micrometers.

[0033] A second aspect of the present invention provides a method for constructing a microfluidic chip, comprising the following steps:

[0034] S1, performing photolithography on a silicon wafer according to a pre-designed pattern, and using polydimethylsiloxane to mold the pattern on the silicon wafer to obtain the polydimethylsiloxane layer;

[0035] S2. Laminating the polydimethylsiloxane layer to the base layer to obtain the microfluidic chip.

[0036] According to some embodiments of the present invention, the pre-designed pattern is designed using AutoCAD or L-edit.

[0037] According to some embodiments of the present invention, the photolithography process includes performing surface plasma and silanization treatment on the silicon wafer.

[0038] According to some embodiments of the present invention, before laminating the polydimethylsiloxane layer to the base layer, the polydimethylsiloxane layer is further cleaned by using a plasma cleaning machine.

[0039] A third aspect of the present invention provides a use of the above-mentioned microfluidic chip in constructing a pancreatic cancer-neural-stromal cell microenvironment model.

[0040] According to some embodiments of the present invention, the application of the above-mentioned microfluidic chip in constructing a pancreatic cancer-neural-stromal cell microenvironment model specifically includes: planting pancreatic cancer cells, neuronal cells and stromal cells in the microchannels of the microfluidic chip for cultivation.

[0041] According to some embodiments of the present invention, the culture temperature is 36.5°C-37.5°C.

[0042] According to some embodiments of the present invention, the culture temperature is 37°C.

[0043] According to some embodiments of the present invention, the volume fraction of carbon dioxide during the culture process is 4%-6%.

[0044] According to some embodiments of the present invention, the volume fraction of carbon dioxide during the culturing process is 5%.

[0045] According to some embodiments of the present invention, the culture medium in the culture process is DMEM culture medium and neuronal culture medium.

[0046] According to some embodiments of the present invention, the neuronal cells are primary dorsal root ganglion neurons; and the stromal cells are pancreatic stellate cells.

[0047] According to some embodiments of the present invention, the microchannel in which the pancreatic cancer cells are seeded is located between two other microchannels.

[0048] According to some embodiments of the present invention, the pancreatic cancer cells, the neuronal cells, and the stromal cells are encapsulated in a hydrogel.

[0049] According to some embodiments of the present invention, the hydrogel includes a hydrogel commonly used in microfluidic chips.

[0050] According to some embodiments of the present invention, the hydrogel also includes a hydrogel obtained based on a decellularized matrix of animal fat.

[0051] A fourth aspect of the present invention provides an application of the above-mentioned microfluidic chip in drug screening.

[0052] According to some embodiments of the present invention, the drug screening includes anti-pancreatic cancer drug screening.

[0053] A fifth aspect of the present invention provides an application of the above-mentioned pancreatic cancer-neural-stromal cell microenvironment model in drug screening.

[0054] According to some embodiments of the present invention, there are at least the following beneficial effects:

[0055] (1) The cell-based microenvironment chip preparation method of the present invention is simple and realizes the co-culture of tumor-neural-stromal cells.

[0056] (2) The present invention realizes the co-construction of pancreatic cancer stromal microenvironment and neural microenvironment through microfluidic technology.

[0057] (3) The pancreatic cancer-neural-stromal cell microenvironment model of the present invention can be used to effectively screen out relevant nanomedicines that can inhibit the growth and metastasis of pancreatic cancer cells, nerve cells, and stellate cells.

[0058] (4) The pancreatic cancer-neural-stromal cell microenvironment model constructed by the present invention improves the efficiency and simulation effect of drug screening, and can perform qualitative and quantitative evaluation of nanomedicines targeting the neural microenvironment and stromal microenvironment. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] The present invention will be further described below with reference to the accompanying drawings and embodiments, in which:

[0060] Figure 1 The figure is a schematic structural diagram of a microfluidic chip of the present invention.

[0061] Figure 2 This is a cell migration diagram based on the pancreatic cancer-neural-stromal cell microenvironment model of the present invention.

[0062] Figure 3 This is a particle size characterization diagram of different nanocarriers based on the pancreatic cancer-neural-stromal cell microenvironment model of the present invention.

[0063] Figure 4 This is a diagram showing the uptake effects of different nanocarriers based on the pancreatic cancer-neural-stromal cell microenvironment model of the present invention.

[0064] Figure 5 This is the therapeutic effect of the nanocarrier loaded with chemotherapy drugs (lipid-PLGA-siRNA) based on the pancreatic cancer-nerve-stromal cell microenvironment model of the present invention. DETAILED DESCRIPTION

[0065] The following will clearly and completely describe the concept and technical effects of the present invention in conjunction with the embodiments to fully understand the purpose, features and effects of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0066] In an embodiment, pancreatic cancer cells and pancreatic stellate cells are purchased from the American type culture collection (ATCC).

[0067] In the embodiments, "plurality" refers to two or more.

[0068] If no specific techniques or conditions are specified in the embodiments, the techniques and conditions described in the literature in this field or the product instructions shall be followed. If the manufacturer of the reagents or instruments used is not indicated, they are all conventional products that can be purchased through regular channels.

[0069] Example 1 Construction of a two-dimensional pancreatic cancer-nerve-stroma microenvironment model

[0070] 1. Extraction and Culture of Primary Dorsal Root Root Neurons

[0071] Isolation and Culture of Primary Dorsal Root Ganglion Neurons (DRG): SD rats within 24 hours of birth were decapitated on ice and their spines removed. The spinal cords were exposed using sterile rongeurs. The dorsal root ganglia located on both sides of the spinal cord were removed and placed in pre-chilled Dulbecco's modified eagle medium (Gibco, Catalog No. 11965092). The DRG tissue was gently rinsed with DMEM and transferred to a 15 mL centrifuge tube. Digested in 10× digestion buffer (Gibco, Catalog No. 15090046) at 37°C for 30 minutes, shaking the tube every 5 minutes to ensure adequate contact between the DRG tissue and the digestion buffer. After digestion, the tubes were terminated with DMEM and gently rinsed three times. Use a sterile pipette to blow the DRG tissue 5-10 times, centrifuge the cell suspension at 1300 rpm for 3 minutes, discard the supernatant, and resuspend the DRG neurons at the bottom in neuronal culture medium (Gibco, catalog number: 21103049), wherein the neuronal culture medium is supplemented with 10% B27 (Gibco, catalog number: A3582801) and 1% double antibody (i.e., penicillin and streptomycin, Gibco, catalog number: 15140122) by mass.

[0072] 2. Preparation of 2D Microfluidic Chips

[0073] The two-dimensional microfluidic chip in the embodiment of the present invention is divided into an upper layer and a lower layer.

[0074] The upper part of the two-dimensional microfluidic chip is a polydimethylsiloxane (PDMS) layer with three rows of parallel microchannels. Each channel is 1 cm long, 500 microns wide, and 200 microns high. The PDMS layer is used to pattern the co-cultured cells. There are smaller microchannels between each parallel microchannel, where the microchannels are 150 microns long, 10 microns wide, and 7 microns high, which are used to build growth connections between cells. The lower part of the two-dimensional microfluidic chip is the base layer, specifically the bottom of the glass slide or culture dish, which constitutes the base for cell culture.

[0075] The PDMS layer on the upper portion of the two-dimensional microfluidic chip is formed using a standard photolithography-based template and soft lithography. Standard photolithography is performed on a silicon wafer; the mask is composed of a chrome plate with a pre-designed pattern using AutoCAD or L-Edit. SU8 photoresist is applied to the silicon wafer using a spin coater and pre-baked after bubbles are eliminated. The baked silicon wafer is covered with the mask and exposed to UV light. The exposed silicon wafer is post-baked, developed with a developer, and stored. The standard photolithography-treated silicon wafer undergoes surface plasma treatment and silanization. PDMS and a crosslinker are mixed in a 10:1 mass ratio and cast onto the silanized silicon wafer. After being placed in a vacuum pump to eliminate bubbles, the entire structure is baked at 80°C for one hour. The resulting cured PDMS layer is perforated, cleaned in a plasma cleaner, and bonded to the bottom of a petri dish.

[0076] 3. Construction of the 2D Model Microenvironment

[0077] The extracted dorsal root ganglion neurons, pancreatic cancer cells, and pancreatic stellate cells were injected into the corresponding areas through the channels on the two-dimensional microfluidic chip for culture at an injection density of 1×10 7 cell / mL, the culture temperature was 37°C, the volume fraction of carbon dioxide during the culture process was 5%, and the culture medium was DMEM culture medium and neuronal culture medium. The pancreatic cancer cells were located in the middle, and the dorsal root neurons and stellate cells were located on both sides. The specific structural model is as follows Figure 1 As shown in the figure, Pancreatic cancer refers to pancreatic cancer cells; Neuron refers to nerve cells; Stromal cell refers to astrocytes.

[0078] Dorsal root ganglion cells, pancreatic cancer cells, and pancreatic stellate cells adhere to and proliferate on a two-dimensional microfluidic chip. Nerve cell axons can grow along the small microchannels to the pancreatic cancer cell area, and stellate cells and pancreatic cancer cells exchange cell secretion factors through the channels. After the three types of cells have grown stably, the upper PDMS module is removed to construct a two-dimensional nerve-pancreatic cancer-stroma microenvironment. Confocal microscopy is used to characterize and observe the migration of different cells, as shown in the following example. Figure 2 shown.

[0079] from Figure 2 It can be seen that with the extension of time, the migration rate of dorsal root neurons to pancreatic cancer cells increased significantly; the migration rate of pancreatic stellate cells to pancreatic cancer cells also increased significantly.

[0080] The two-dimensional model microenvironment can precisely control cell patterning to form a neural-tumor-stromal cell network. In addition, the two-dimensional level can more clearly observe cell migration, proliferation and other growth conditions, making it easier to quantitatively evaluate various parameters.

[0081] Example 2 Construction of a three-dimensional pancreatic cancer-neural-stromal cell microenvironment model

[0082] Using 3D printing technology, nerve cells, pancreatic cancer cells, and pancreatic stellate cells were encapsulated in a hydrogel and arranged in parallel. From left to right, the overall model consists of a stellate cell region (500 microns wide), a hydrogel region (400 microns wide), a cancer cell region (400 microns wide), a hydrogel region (400 microns wide), and a nerve cell region (500 microns wide). The microporous structure of the hydrogel region ensures the mutual transmission of cytokines, drugs, etc.

[0083] The three-dimensional model microenvironment is based on the two-dimensional model microenvironment combined with hydrogel to form a physical and chemical microenvironment similar to that in vivo, providing similar microcomponents as in vivo for cell growth, and can further restore the pancreatic cancer microenvironment state in vivo.

[0084] Example 3 Application of three-dimensional model microenvironment in nanodrug screening

[0085] 1. Construction of a nanodrug library based on a three-dimensional model microenvironment

[0086] Based on the design and synthesis of biomedical nanomaterials accumulated by our laboratory, combined with the synthesis methods of related technologies, we will design and synthesize different types of nanomaterials, including inorganic nanoparticles (such as gold nanoclusters), cationic liposomes (such as phospholipids), solid lipid nanoparticles (such as lipid-PLGA), etc.

[0087] In this embodiment of the present invention, microfluidic technology is used to control the synthesis of solid lipid nanoparticles (lipid-PLGA) with varying particle sizes, surface charges, hardnesses, and surface modifications to construct a library of neuro-related nanodrugs. Furthermore, gene editing using NGF-siRNA and BDNF-siRNA, which have been screened to inhibit nerve signaling pathways, or the related CRISPR / Cas9 system is used to disrupt tumor-nerve and tumor-stroma signaling pathways.

[0088] Furthermore, by combining physical and chemical characterization methods to optimize synthesis conditions, a stable and effective library of neuro-related nanomedicines was screened. The present invention uses microfluidic chip technology to control parameters such as the flow rate, ratio, and starting concentration of different raw materials to synthesize solid lipid nanoparticles (lipid-PLGA) with varying particle sizes, surface charges, or hardness.

[0089] 2. Characterization of Neuro-Related Nanodrug Libraries for High-Throughput Drug Screening

[0090] The synthesized three-dimensional model microenvironment-related nanodrug library was characterized by a series of physical and chemical methods to determine its composition, spatial morphology, and drug loading. In this example, dynamic light scattering (DLS) was used to characterize the hydration kinetic diameter and dispersibility of the synthesized nanomaterials. Figure 3 shown.

[0091] from Figure 3 The results show that the three nanocarriers (lipid, PLGA, and lipid-PLGA) have relatively uniform sizes. The lipid nanocarrier has the highest size distribution percentage at 30 nm; the PLGA nanocarrier has the highest size distribution percentage at around 60 nm; and the lipid-PLGA nanocarrier has the highest size distribution percentage at 70 nm. This indicates that the pancreatic cancer-neural-stromal cell microenvironment chip of the present invention can be used to effectively screen for the optimal particle size of different nanocarriers.

[0092] 3. Screening of Nanodrug Libraries Based on High-Throughput Drug Screening Platforms

[0093] Dorsal root ganglion cells, pancreatic cancer cells, and pancreatic stellate cells were used as research objects. The present invention constructed a co-culture platform based on the dorsal root ganglion cell-pancreatic cancer cell-stromal cell microenvironment model to evaluate the uptake of related nanocarriers (lipid, PLGA, and lipid-PLGA) in different cells.

[0094] The fluorescence distribution of fluorescently labeled nanocarriers (lipid, PLGA, and lipid-PLGA) in cells was determined by laser confocal microscopy. Figure 4 shown.

[0095] from Figure 4 As can be seen in the results, compared with the control, PLGA, and lipid groups, pancreatic cancer cells (Panc-1), pancreatic stellate cells (PSC), and dorsal root ganglion cells (neurons) showed the highest uptake of lipid-PLGA. This indicates that the neural cell-pancreatic cancer cell-stromal cell microenvironment model constructed using the microfluidic chip of the present invention can effectively assess the cellular uptake of relevant nanocarriers (lipid, PLGA, and lipid-PLGA) and screen for the optimal nanodrug carrier.

[0096] In addition, through the neural cell-pancreatic cancer cell-stromal cell co-culture platform, confocal microscopy was used to further evaluate the effects of related nanomedicines (such as lipid-PLGA-siRNA, where siRNA includes NGF siRNA and BDNF siRNA targeting the neural microenvironment of pancreatic cancer, and PDGF siRNA and TGFβ siRNA targeting the stromal microenvironment of pancreatic cancer) on the proliferation and migration levels of tumor cells and stellate cells, as well as the growth status of neural cells, and the migration of cells. The results are as follows Figure 5 shown.

[0097] from Figure 5 It can be seen that compared with the migration of pancreatic cancer cells and neurons in the control group, there was no significant change in the ability of pancreatic cancer cells to migrate toward neurites in the lipid-PLGA-PDGF siRNA group; there was no significant change in the ability of pancreatic cancer cells to migrate toward neurites in the lipid-PLGA-TGFβ siRNA group; the ability of pancreatic cancer cells to migrate toward neurites in the lipid-PLGA-NGF siRNA group was significantly decreased; and the ability of pancreatic cancer cells to migrate toward neurites in the lipid-PLGA-BDNF siRNA group was significantly decreased.

[0098] Compared with the migration of pancreatic cancer cells and stellate cells in the control group, the mutual migration ability between pancreatic cancer cells and pancreatic stellate cells in the lipid-PLGA-PDGF siRNA group was significantly decreased; the mutual migration ability between pancreatic cancer cells and pancreatic stellate cells in the lipid-PLGA-TGFβ siRNA group was significantly decreased; the mutual migration ability between pancreatic cancer cells and pancreatic stellate cells in the lipid-PLGA-NGF siRNA group was slightly decreased; and the mutual migration ability between pancreatic cancer cells and pancreatic stellate cells in the lipid-PLGA-BDNF siRNA group was slightly decreased.

[0099] Through the above scheme, it can be seen that the pancreatic cancer-neural-stromal cell microenvironment model of the present invention can effectively screen relevant nanomedicines that can inhibit the growth and metastasis of pancreatic cancer cells, nerve cells, and stellate cells.

[0100] In summary, the present invention constructs a microfluidic chip, the preparation method of which is simple, and the present invention realizes the co-culture of three types of cells: nerves, pancreatic cancer, and stellate cells, and simultaneously establishes two pancreatic cancer microenvironments (including matrix microenvironment and neural microenvironment). Secondly, starting from the connective tissue and neural microenvironment, the present invention can also combine CRISPR / Cas9 gene technology and innovative nanocarriers to develop two-dimensional and three-dimensional pancreatic cancer-nerve-stromal cell microenvironments based on microfluidic technology. On the one hand, the present invention realizes the co-construction of pancreatic cancer matrix microenvironment and neural microenvironment through microfluidic technology, and simulates two microenvironments of pancreatic cancer on one platform at the same time. On the other hand, the present invention improves the efficiency and simulation effect of drug screening through microfluidic technology, and can perform qualitative and quantitative evaluation of nanomedicines targeting neural microenvironment and matrix microenvironment; and the pancreatic cancer research models and technical means in the related art cannot achieve the above two points.

[0101] While the embodiments of the present invention have been described in detail above with reference to the accompanying drawings, the present invention is not limited to the embodiments described above. Various modifications may be made within the scope of knowledge possessed by a person skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof may be combined with one another unless there is a conflict.

Claims

1. Application of a microfluidic chip in constructing a pancreatic cancer-neural-stromal cell microenvironment model, characterized in that: include: Pancreatic cancer cells, primary dorsal root ganglion neurons, and stromal cells were seeded and cultured in the microchannels of a microfluidic chip. Wherein, the stromal cells are pancreatic stellate cells; The microfluidic chip includes a polydimethylsiloxane layer and a base layer; The polydimethylsiloxane layer comprises three microchannels, and the microchannel where the pancreatic cancer cells are planted is located in the middle of the other two microchannels; The microchannel is provided with a hole connected to the outside world; The microchannels are connected by a plurality of parallel microchannels, wherein the length of the microchannels is 100-200 microns, the width is 8-12 microns, and the height is 5-10 microns; The base layer is at least one of a glass base layer, a polydimethylsiloxane base layer or a polystyrene base layer.

2. The use according to claim 1, characterized in that The length of the microchannel is 800-1200 microns.

3. The use according to claim 2, characterized in that The width of the microchannel is 400-600 micrometers, and the height of the microchannel is 150-250 micrometers.

4. The use according to claim 1, characterized in that The method for constructing the microfluidic chip comprises the following steps: S1, performing photolithography on a silicon wafer according to a pre-designed pattern, and using polydimethylsiloxane to mold the pattern on the silicon wafer to obtain the polydimethylsiloxane layer; S2. Laminating the polydimethylsiloxane layer to the base layer to obtain the microfluidic chip.

5. The use according to any one of claims 1 to 4, characterized in that: The pancreatic cancer cells, the neuronal cells and the matrix cells are encapsulated in a hydrogel and cultured.

6. Application of a microfluidic chip in drug screening, characterized in that: The microfluidic chip includes a polydimethylsiloxane layer and a base layer; The polydimethylsiloxane layer comprises three microchannels, and pancreatic cancer cells, primary dorsal root ganglion neurons, and stromal cells are respectively planted in the microchannels of the microfluidic chip for culture, with the microchannel in which the pancreatic cancer cells are planted being located between the other two microchannels; the stromal cells are pancreatic stellate cells; The microchannel is provided with a hole connected to the outside world; The microchannels are connected by a plurality of parallel microchannels, wherein the length of the microchannels is 100-200 microns, the width is 8-12 microns, and the height is 5-10 microns; The base layer is at least one of a glass base layer, a polydimethylsiloxane base layer or a polystyrene base layer.

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