Microfluidic device and application thereof

Abstract

The invention relates to the field of basic medicine, in particular to a microfluidic device and application thereof. Specifically, the invention provides a microfluidic device characterized by three annular micro-column arrays, which can effectively project three-dimensional tumor sphere features onto a two-dimensional plane without a complex pipeline system. The device facilitates the formation of a microenvironment having different layers: a peripheral proliferative layer, an intermediate stationary layer, and an inner necrotic core. By means of the microfluidic device, continuous pH, oxygen and hardness gradients can be effectively established, and classic gradient layer distribution observed in tumor balls can be copied.

Description

Technical Field

[0001] The present invention relates to the field of basic medicine, and in particular to a microfluidic device and applications thereof. Background Art

[0002] Compared with traditional two-dimensional (2D) culture systems, three-dimensional (3D) tumor spheroid models can more realistically reflect the physiological characteristics and complexity of in vivo tumors. Tumor spheroids contain basic spatial gradient distribution characteristics, which is also one of the basic hallmarks of solid tumors. This spatial characteristic is formed by a complex interaction between vascular permeability and the strong proliferation ability of tumor cells, resulting in a gradient environment from the proximal to the distal regions of the blood vessels. The importance of the gradient microenvironment has been considered to play a key role in the complex processes of tumorigenesis, invasion, metastasis, and acquisition of drug resistance. Similarly, as the spheroid grows, the distance from the external microenvironment increases, leading to the establishment of chemical gradients (pH, oxygen, glucose, ATP distribution, lactate accumulation, etc.), thereby hindering the metabolic activities of tumor cells in the internal space. At the same time, different regions with different proliferation abilities appear, dividing the tumor spheroids into a peripheral proliferating layer, an intermediate quiescent layer, and an inner necrotic core. Thanks to the remarkable similarities between tumor spheres and solid tumors, research progress based on tumor sphere models has greatly enhanced our understanding of tumor biology and contributed to therapeutic advances.

[0003] However, the traditional tumor sphere model exhibits two inherent deficiencies, size and shape. Studies have shown that as the tumor sphere grows, the solid stress caused by cell growth and proliferation imposes restrictions on its growth, and its upper limit in diameter is estimated to be around 1000μm. At this scale, downstream analysis (such as paraffin embedding and section preparation) becomes challenging, especially extracting cells from different proliferation areas for molecular mechanism analysis becomes extremely difficult. The second challenge is the spherical structure of the tumor sphere. In addition to optical sectioning observation using an optical microscope, it is difficult to obtain the internal characteristics of the tumor sphere, especially real-time monitoring, using a traditional inverted fluorescence microscope.

[0004] Advances in microfluidic technology have addressed several challenges faced by traditional tumor sphere models. Studies have reported the development of different microfluidic devices, mainly using flow-based or diffusion-based strategies to simulate and control the gradient properties of tumor spheres or solid tumors (such as oxygen, pH, cell viability, etc.). Moreover, most microfluidic platforms use transparent substrates, which simplifies the real-time observation of different microenvironmental regions. However, there has been no report on a device that can establish a gradient spatial distribution feature similar to that of tumor spheres, especially to achieve a radial distribution from the peripheral layer to the inner core, and subsequently allow the extraction of cellular components from different regions for downstream molecular mechanism analysis, such as histological staining and qPCR analysis or sequencing. Summary of the invention

[0005] In order to solve the above technical problems, we have developed a novel microfluidic device (hereinafter also referred to as a device or chip in the embodiments) that can effectively project the three-dimensional tumor sphere features onto a two-dimensional plane without the need for a complex piping system. The device helps to form a spherical microenvironment with different layers: a peripheral proliferating layer, an intermediate quiescent layer, and an inner necrotic core. The microfluidic device of the present application can effectively establish a continuous pH, oxygen, and stiffness gradient, replicating the classic gradient layer distribution observed in tumor spheroids.

[0006] The device features three annular arrays of micropillars designed to confine a mixture of cells and gelatin methacrylate (GelMA) to a central region. This design replicates the conditions required to form a tumor spherical microenvironment, allowing nutrients and oxygen to diffuse from the periphery to the inner core. A gradient distribution of cell proliferation status, pH, oxygen levels, and stiffness characteristics is established on the chip.

[0007] After in vitro culture, the mixture of tumor cells and GelMA has completely fused together, referred to as "cell aggregates" in this application. The cell aggregates on the device can be easily released, and cells can be easily retrieved from different gradient areas for downstream molecular analysis. Compared with existing methods, the microfluidic device of this application is more friendly and comprehensive in replicating the tumor sphere microenvironment and has good scalability. It can be used as a versatile tool to study gradient environments related to tumor biology research, including chemotaxis, cell-cell interactions, drug resistance mechanisms, and immune infiltration.

[0008] Specifically, this application provides the following technical solutions:

[0009] In the first aspect, the present invention provides a microfluidic device, comprising a substrate and a capping layer, the capping layer having a recessed area, the recessed area having three layers of microcolumns arranged in a concentric circle shape, the height of the microcolumns being the same as the depth of the recessed area, the diameter of the largest circle in the concentric circles being 1000-10000 μm, the distance between microcolumns in the same layer being 100 μm, and the intervals between different layers being 100 μm, the concentric circles having a perforation at the center thereof that penetrates the capping layer.

[0010] Preferably, the depth of the recessed area is 100-1000 μm, specifically including 100 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm and 1000 μm.

[0011] Most preferably, the depth of the recessed area is 300 μm, and the height of the microcolumn is also 300 μm.

[0012] Preferably, the diameter of the largest circle among the concentric circles includes 1000 μm, 2000 μm, 3000 μm, 4000 μm, 5000 μm, 6000 μm, 7000 μm, 8000 μm, 9000 μm and 10000 μm.

[0013] Most preferably, the diameter of the largest circle among the concentric circles is 6000 μm.

[0014] Preferably, the length of the cross section of the microcolumn is 100 μm and the width is 50 μm, wherein the length of the side perpendicular to the radius of the circle is called the length, and the length of the side parallel to the radius of the circle is called the width.

[0015] Preferably, one or more columns with a height of 300 μm can be designed in the area surrounded by the concentric circles to maintain the spatial structure. In a specific embodiment of the present invention, 12 columns are set for experimental verification.

[0016] Preferably, the diameter of the perforations is 1 mm.

[0017] Preferably, the recessed area is in any shape.

[0018] Preferably, the recessed area is circular.

[0019] More preferably, the center of the concave area is located at the same position as the center of the three layers of microcolumns arranged in a concentric circle shape.

[0020] More preferably, the diameter of the recessed area is adjusted according to the diameter of the largest circle among the concentric circles. When the diameter of the largest circle among the concentric circles is 6000 μm, the diameter of the recessed area is preferably 12 mm.

[0021] Preferably, the cover layer has one or more holes, and the holes are connected to the recessed area.

[0022] Preferably, the hole may be of any shape, preferably circular (approximately circular, with the area overlapping the recessed area forming a gap).

[0023] Preferably, the thickness of the capping layer is greater than the depth of the recessed area.

[0024] Preferably, the base plate has a diameter of 34 mm so that it can be placed in a conventional 6-well plate for compatibility and batch operation.

[0025] Preferably, the substrate is transparent.

[0026] Preferably, the substrate may be made of silicon, glass or ceramic.

[0027] Preferably, the substrate is made of glass.

[0028] Preferably, the material of the capping layer is selected from polydimethylsiloxane (PDMS), acrylonitrile-butadiene-styrene copolymer, polycarbonate (PC), polymethyl methacrylate (PMMA), polyurethane, polyethylene, polypropylene, polymethylpentene, polytetrafluoroethylene (PTFE), polyvinyl chloride (PVC), cyclic polyolefin copolymers (Cyclic Olefin Copolymers, COC), polyvinylidene fluoride, polystyrene, polysulfone, nylon, styrene-acrylic acid copolymer, natural rubber, polyisoprene, butyl rubber, halogenated butyl rubber, polybutadiene, styrene-butadiene rubber, nitrile rubber, chloroprene rubber, ethylene-propylene rubber, epichlorohydrin rubber, polyacrylate rubber, silicone rubber, fluorosilicone rubber, fluorine-containing elastomer (FKM), perfluoroelastomer (FFKM), ethylene-vinyl acetate, resilin, elastin, polyimide, phenolic resin, or a mixture of any two or more thereof.

[0029] Preferably, the material of the capping layer is selected from polydimethylsiloxane (PDMS).

[0030] Specifically, when in use, the capping layer is placed on the substrate, the recessed area and the substrate form a cavity (central cell chamber), and the cell-GelMA mixture containing cells is loaded through the perforation (cell loading entrance). The three-layer concentric circle structure of the capping layer arranges the microcolumns to prevent cell penetration and loss; cell culture medium is added to the holes, the cell culture medium can penetrate into the cavity and provide the cell culture with the components required for cell growth, forming a gradient characteristic similar to that of a tumor sphere, including gradient characteristics of pH, hypoxia and hardness.

[0031] On the other hand, the present invention provides the use of the above microfluidic device in simulating the gradient microenvironment in tumor spheres.

[0032] On the other hand, the present invention provides a method for simulating a gradient microenvironment in a tumor sphere in a 2D plane, the method comprising assembling a substrate and a capping layer of the aforementioned microfluidic device, adding a mixture of cells and GelMA into a cavity formed by the substrate and the capping layer through perforations on the capping layer, adding cell culture medium into the holes of the capping layer, placing the mixture and cell culture medium under standard cell culture conditions after addition, and replacing the culture medium regularly.

[0033] Preferably, the mixture covers (fills) the cavity.

[0034] Preferably, after the mixture is added to the cavity, a thin sheet is used to cover (close) the perforation, which can be a 9 mm cover glass.

[0035] Preferably, the number of cells in the mixture is less than 1×10 6 cells / 10μL GelMA.

[0036] Preferably, the ratio of GelMA to cells in the mixture can be 1-10×10 5 cells / 10μLGelMA, including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10×10 5 cells / 10μL GelMA.

[0037] Preferably, the standard cell culture conditions include, for example, culturing in a humidified incubator at 37° C. and 5% CO 2 .

[0038] Preferably, the microfluidic device is placed in a container containing a small amount of water (sterile water) to prevent excessive evaporation, and can be placed in a 6-well plate, and the gaps outside the wells of the 6-well plate need to be filled with sterile water.

[0039] Preferably, the frequency of changing the culture medium and the total culture time can be determined according to the cell characteristics and growth status, for example, changing the medium every two days for a total of 1-10 or more days of culture.

[0040] Preferably, the cells include any cells derived from humans, gorillas, monkeys, horses, cows, sheep, pigs, donkeys, camels, dogs, rabbits, cats, rats, mice, fish, birds or insects.

[0041] Preferably, the cells include primary cells, tissue-derived stem cells, tissue-derived precursor cells, induced pluripotent stem cells, differentiated cells, transdifferentiation cells, cells obtained by cell engineering, tumor stem cells, tumor cells isolated from tumor tissue, tumor-associated fibroblasts, stromal cells, immune cells, cell strains, cell lines, insect cells, cell clumps, tissue clumps, and in vitro cultured organs.

[0042] Preferably, the culture medium can be any culture medium suitable for target cells, such as one or more of DMEM / F12 cell culture medium, William's E cell culture medium, Neurobasal Medium cell culture medium, MEM cell culture medium, DMEM cell culture medium, 1640RPMI cell culture medium, or F12 cell culture medium.

[0043] In another aspect, the present invention provides a method for preparing a cell slice having a gradient microenvironment, the method comprising the steps of culturing cells and fixing cells,

[0044] The cell culture step is: assembling the substrate and the cover layer of the aforementioned microfluidic device, adding the mixture of cells and GelMA into the cavity formed by the substrate and the cover layer through the perforations on the cover layer, adding cell culture medium into the holes of the cover layer, placing under standard cell culture conditions after adding the mixture and the cell culture medium, and replacing the culture medium regularly;

[0045] The fixing step is: separating the substrate and the cover layer, and adding a fixing solution to the cells remaining on the substrate for fixing.

[0046] Preferably, the slice can be segmented so as to conduct targeted observation and detection of cells in specific areas therein.

[0047] Preferably, the fixative comprises 4% paraformaldehyde.

[0048] Preferably, the cell sections can be used for conventional detection, such as H&E staining, fluorescent staining, PCR detection, etc.

[0049] In another aspect, the present invention provides a cell slice prepared by the above method.

[0050] Preferably, the cell characteristics in the cell slice are distributed in a gradient, and the cell characteristics include proliferation state, pH value, oxygen level and hardness characteristics. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1Schematic diagrams: A is a schematic diagram of the gradient microenvironment characteristics of the tumor microenvironment (TME), B is a schematic diagram of the cell loading and incubation process, C is a schematic diagram of the structure of the PDMS layer, and D is a schematic diagram of the manufacturing and assembly.

[0052] Figure 2 These are the results of observing the gradient microenvironment in a two-dimensional plane: A is the gradient activity distribution of tumor cells on the chip, B is the relative diameter comparison of cell spheres and cell aggregates of different sizes in the suspension culture plate, C is the fluorescence image of traditional tumor spheres under an ordinary microscope, and D is the cell fluorescence intensity distribution result diagram on the chip.

[0053] Figure 3 The fluorescence images obtained by culturing with different initial cell densities: A is the result of spherical gradient microenvironment formed by different initial cell densities, scale bar = 1000 μm, B is Figure 3 A. Statistical results of fluorescence intensity.

[0054] Figure 4 The figures are the detection results of pH, hypoxia and hardness distribution characteristics: A is the detection result of pH probe pHrodo Red AM, B is the detection result of pH probe BCECF AM, C is the detection result of hypoxia probe Image-iT Green (RFU: relative fluorescence unit), D is a schematic diagram of the method of analyzing the hardness distribution characteristics of cell aggregates from the outside to the inside by AFM after disassembling the device (****p<0.0001).

[0055] Figure 5 These are the rhodamine permeation monitoring result diagrams of Chip_gel. A is the permeation process result diagram, B is the time series quantitative result diagram, and C is the permeability result diagram.

[0056] Figure 6 These are the rhodamine permeation monitoring result diagrams for Chip_day0. A is the permeation process result diagram, B is the time series quantitative result diagram, and C is the permeability result diagram.

[0057] Figure 7 These are the rhodamine permeation monitoring results of the chip with a fully established gradient microenvironment. A is the permeation process result diagram, B is the time series quantitative result diagram, and C is the permeability result diagram.

[0058] Figure 8 Schematic diagram of the histological sectioning method of cell aggregates and the results of H&E staining and immunofluorescence staining: A is a schematic diagram, B is the result of H&E staining, with scale bars of 500μm and 100μm, respectively, and C is the result of immunofluorescence staining, with scale bars of 1000μm and 50μm, respectively.

[0059] Fig. 9This is a physical picture of the disassembly process of the microfluidic device of this application.

[0060] Fig.10 This is a graph showing the gene expression detection results of cells in different regions. DETAILED DESCRIPTION

[0061] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes based on the technical solution and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

[0062] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0063] Example 1. Design and manufacture of microfluidic device (chip)

[0064] The microfluidic device of the present application is designed with three layers of concentric columns. The distance between the columns on each concentric circle is 100 μm. In addition, the radius difference between the layers with columns is also 100 μm. The cross-sectional dimensions of the columns are 100 mm long and 50 mm wide. The chip is designed with sparse columns inside to support its spatial structure. The mold of the chip is made using standard photolithography procedures.

[0065] The specific manufacturing method is: use standard photolithography process to make the mold, spin-coat MicroChem's SU-8 2150 resist onto the silicon wafer at 500 rpm for 10 seconds, and then spin at 2000 rpm for 30 seconds. Then, soft bake, 250 mJ / cm 2 After 365nm ultraviolet exposure and post-baking, the master of the device is obtained and placed in a culture dish for use. After mixing the PDMS resin and the curing agent in a ratio of 10:1, vacuum degassing is performed, and the mixed PDMS is poured into the culture dish where the master is placed, about 2-5mm above the surface of the master. After degassing, it is placed in a 70℃ oven for curing for 2h. After cooling, the PDMS layer is carefully peeled off from the master. According to the design, the required parts of this device are divided to obtain the capping layer. Punch holes at the corresponding positions of the capping layer, and then evenly apply a thin layer of newly configured PDMS on the bottom of the capping layer for bonding the glass substrate. The capping layer is placed vertically on the glass substrate and baked briefly at 80℃ for 15 minutes to combine the capping layer with the glass substrate. This assembly method ensures that the device does not leak while also facilitating the subsequent disassembly of the device.

[0066] The design of the chip is inspired by the formation mechanism of tumor spheres, which are closed spherical structures that allow nutrients to diffuse from the periphery to the core ( Figure 1 A). To enhance user-friendliness and increase operational throughput, we chose a 34 mm diameter round glass substrate as the substrate for the microfluidic device. In addition, the culture medium was perfused through the reservoirs at both ends of the device, without the need for an external flow system. This design ensures that our device is compatible with standard 6-well plates and can be seamlessly integrated with common microscope platforms, facilitating batch operation and real-time monitoring ( Figure 1 B).

[0067] The central region of the device is the cell chamber, which is 6 mm in diameter, 300 μm deep, and has a 1 mm diameter perforation for loading the cell-GelMA mixture. The central cell chamber is surrounded by three concentric arrays of micropillars, which are 100 μm long and 50 μm wide. The micropillars in each array are spaced 100 μm apart. This design is intended to prevent accidental leakage of the cell-GelMA mixture during the initial loading step while allowing nutrient penetration ( Figure 1 C).

[0068] Figure 1 D illustrates the relative dimensions of the assembled microfluidic device and the introduction of crystal violet solution into the device, with the central cell compartment surrounded by crystal violet solution to show the distribution of the culture medium.

[0069] Example 2: Detection of gradient microenvironment

[0070] SK-Hep-1 wild-type cell line and GFP-transfected SK-Hep-1 cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Invitrogen) supplemented with 10% fetal bovine serum (FBS, Invitrogen) and 1% penicillin-streptomycin (Invitrogen). The cells or chips loaded with cells were cultured in a humidified incubator at 37°C and 5% CO2.

[0071] A predefined hole with a diameter of 1 mm was made in the central area of ​​the microfluidic device prepared in Example 1. Cells were dissociated with enzymes and centrifuged to obtain cell pellets. The cell pellets were mixed with 10% GelMA-60 (the resulting mixture is called a mixture of cells and gelatin methacrylate, also referred to as cell-GelMA) and introduced into the chip through the predefined hole until they completely covered the area surrounded by the concentric columns. The cells were then electrophoresed at 405 nm with a power of 60 mW cm -2The cell-GelMA was solidified by briefly irradiating with a power density of 100 nm for 15 seconds. After solidification, the central area of ​​the chip surface was covered with a thin cover glass with a diameter of 9 mm to seal the holes and prevent oxygen diffusion. The chip was then supplemented with DMEM medium through the circular holes at both ends of the chip and then placed in a 6-well plate under standard cell culture conditions ( Figure 1 ). It should be noted that the gaps outside the 6-well plate wells need to be filled with sterile water to prevent excessive evaporation of the culture medium in the chip, and the culture medium should be replaced every two days to maintain optimal growth conditions.

[0072] SK-Hep1 cells transfected with GFP established a gradient microenvironment on the chip, which showed that the chip successfully replicated the gradient of cell viability, which was very similar to the classic structure of tumor spheres. This structure includes a peripheral area with the highest cell proliferation, followed by an intermediate static layer with gradually decreasing cell activity, and finally culminated in a central apoptotic area with almost no cells ( Figure 2 A).

[0073] Unlike previously reported microfluidic platforms where the cell loading inlet is usually located at the edge of the cell chamber, we strategically positioned the cell loading inlet at the center. This innovative design facilitates the establishment of a circular, closed, and continuous gradient microenvironment. The relative sizes of the cell aggregates were directly compared with those of tumor spheres cultured in suspension dishes with diameters ranging from 100 μm to 900 μm, indicating that the device effectively addresses the inherent size limitations ( Figure 2 B). The diameter of the cell aggregate is about 6 mm, which provides ample operating space for subsequent histological sectioning and cell extraction analysis. In addition, the chamber size can be flexibly adjusted according to specific experimental requirements.

[0074] This method greatly facilitates the two-dimensional plane observation of cell contours within the internal area of ​​the tumor sphere by projecting the three-dimensional tumor sphere features onto a two-dimensional plane. Figure 2 CD).

[0075] Example 3: Effect of initial cell density on the formation of gradient microenvironment

[0076] In the microfluidic device prepared in Example 1, the formation mechanism of the cell aggregate microenvironment gradient mainly depends on the natural osmotic gradient of nutrients from the periphery to the interior, combined with the cell metabolic process. Therefore, the initial cell density plays a vital role in determining the final result. In this application, we evaluated the effect of initial cell density on the establishment of a spherical gradient microenvironment.

[0077] like Figure 3 As shown in A, 1×10 6 cells / 10μL GelMA was loaded with cell-GelMA, and obvious gradient characteristics began to appear from the 4th day.5 cells / 10μL and 2.5×10 5 cells / 10μL decreased, and the gradient characteristics began to appear from the 6th and 14th days, respectively. The initial cell density was lower than 2.5×10 5 The cells / 10μL chip had difficulty forming a gradient microenvironment within the 14 days of monitoring, similar to the traditional 3D spheroid culture microenvironment. The trend of fluorescence intensity over time clearly showed that as the cell density decreased, the nutrient consumption rate gradually fell below the diffusion rate, making it difficult to establish a gradient environment ( Figure 3 B).

[0078] It should be noted that the initial cell density cannot be increased indefinitely, because 1×10 6 The cells filled up nearly 10μL GelMA. Further increasing the cell density would make the cell-GelMA too viscous to load, or the proportion of GelMA was too low to make the cell-GelMA difficult to solidify into a gel. As the gradient environment was established, the cells remaining in the central loading inlet gradually died, which had the least impact on the final microenvironment phenotype.

[0079] Example 4: Detection of characteristics of microenvironment gradient distribution

[0080] Wild-type SK-Hep1 cells were first trypsinized, washed with PBS, and then incubated with the corresponding dyes. Cells labeled with probes were washed once with PBS, loaded into the chip as described previously, and incubated in an incubator. After 8 h of incubation, cells on the chip were analyzed using a Nikon Eclipse Ti2 inverted microscope.

[0081] To evaluate the pH and oxygen gradient distribution within the device, we used commercially available dyes pHrodo Red AM (P35372, Thermo Fisher) and BCECF AM (S1006, Beyotime) for pH analysis, and Image-iT Green Hypoxia Reagent (I14833, Thermo Fisher) for oxygen analysis. These compounds were dissolved in dimethyl sulfoxide (DMSO) as recommended by the supplier. The staining procedure was performed according to the manufacturer's instructions. The pH distribution state was first evaluated using pHrodo Red AM. Figure 4A shows a gradient-increasing fluorescence signal from the periphery to the central area, which means that the central area exhibits a stronger acidic microenvironment compared to the periphery. Due to the cell loading mode mentioned above, residual cells at the loading inlet can cause a bright spot in the center. Although this does not affect the formation of the final microenvironment, in order to eliminate potential nonspecific interference with the results, we introduced another probe, BCECF AM, to further confirm the pH distribution curve along the chip. The fluorescence generation mechanism of BCECF AM is opposite to that of pHrodo Red AM, and cells labeled with BCECF AM show lower signal intensity in a lower pH microenvironment. The final results are consistent. BCECF AM staining confirmed the gradient decrease of the signal from the periphery to the central area, confirming the low pH level in the central area ( Figure 4 B). In addition, we observed that the nonspecific fluorescence within the cell loading inlet was confined to a very small area, and its impact on the surrounding signals was negligible. Next, the cells were stained with the hypoxia probe Image-iT Green, confirming that the cells in the central area were under high levels of hypoxia and gradually eased toward the outer areas ( Figure 4 C).

[0082] In addition, the PDMS layer (capping layer) can be removed from the chip to expose the cell aggregates to characterize the stiffness distribution curve, which is an operation that is difficult to achieve with traditional tumor sphere models. Refer to the previously reported method (Nanomechanical characterization of exosomes and concomitant nanoparticles from blood plasma by PeakForce AFM in liquid. Biochim Biophys Acta Gen Subj, 2022.1866(7): p.130139.) to analyze the chip hardness curve. In short, on the seventh day, when the gradient microenvironment was formed and stabilized, the chip was removed from the 6-well plate and the PDMS layer of the chip was carefully removed. The cell aggregates on the glass slide were immersed in PBS and AFM measurements were performed in contact mode using a Bioscope Catalyst atomic force microscope (Bruker Instruments, USA) ( Figure 4 D), the radius of the pyramidal tip was 20 nm. The deflection sensitivity and spring constant of each probe were calibrated on a glass slide before each measurement. The Poisson's ratio was set to 0.3, and the Sneddon force curve fitting model was used to calculate the Young's modulus.

[0083] The experiments showed that the hardness curve in the central area is significantly higher ( Figure 4D). Hypoxia and pH properties inside tumor spheres have been widely reported for quite some time. Current studies on tumor sphere stiffness mainly focus on overall stiffness. To our knowledge, there are limited reports on the characterization of stiffness distribution inside tumor spheres due to the natural limitation caused by their closed spherical structure. However, it has been reported that hypoxia can induce increased tumor stiffness by upregulating multiple collagen modifying enzymes. This could explain our observed results.

[0084] Example 5: Real-time monitoring of the infiltration process

[0085] The glass substrate and circular cell chamber design facilitate real-time observation of the permeation process in the gradient microenvironment chip. This application uses rhodamine to characterize its permeation dynamics in three different microenvironments.

[0086] The initial chip type consisted of only GelMA without cells loaded (Chip_GelMA), while the second chip type had just completed loading GelMA and cells (Chip_Day0). The third chip type had a stable gradient microenvironment that was cultured for 10 days (Chip_env). Rhodamine was diluted into DMEM to a final concentration of 10 μM. The chip was washed three times with DMEM containing rhodamine to replace the original culture medium. The device was then placed under a Nikon Eclipse Ti-E live cell imaging system for observation. For Chip_GelMA, images were captured every 10 minutes over a 2-hour period. Chip_Day0 was monitored every 20 minutes over a 12-hour period. For Chip_env, images were recorded once at 10-minute intervals for 18 hours.

[0087] We first evaluated the permeability of rhodamine on Chip_gel, which refers to a chip loaded with GelMA but without cells ( Figure 5 ) and was observed to penetrate effortlessly into the inner region of the chip within about 2 hours ( Figure 5 A and 5B), the average diffusion rate was about 20.7±7.3μm / min ( Figure 5 C).

[0088] Subsequently, we plotted the permeation curve of rhodamine on Chip_day0, where “Chip_day0” represents the chip loaded with the GelMA-cell mixture before in vitro culture ( Figure 6 ), in which case the tumor microenvironment within the chip has not yet been established. It is clear that in the presence of cells, the permeability of rhodamine is significantly reduced ( Figure 6 A and 6B), the penetration rate is about 4.0±2.4μm / min ( Figure 6 C).

[0089] Furthermore, the introduction of rhodamine into the fully established gradient microenvironment showed a prolonged permeation process even after an extended time of more than 24 h. External rhodamine had difficulty penetrating into the central region ( Figure 7 A and 7B), the overall diffusion rate was significantly reduced to 1.53±0.2μm / min ( Figure 7 C) Towards the end of the monitoring period, the penetration rate had reached almost zero.

[0090] Unlike previously reported microfluidic devices, the chip of this application places the cell entrance in the central area, so that the edge of our cell cavity forms a continuous circular contour, thereby realizing a concentric diffusion process from the uninterrupted edge to the center, effectively reproducing the permeation characteristics of substances entering tumor spheres. Combining the permeation curves of rhodamine in three different types of microenvironment chips, it can be seen that in chips that form gradient microenvironments, it is difficult for external substances to penetrate into the chip, which eventually leads to the formation of new concentration gradient characteristics inside the chip. This permeation difference is also similar to the previously reported phenomenon that drugs have difficulty penetrating apoptotic areas. Both results reveal the potential of our platform in real-time observation of drug permeation processes and exploration of strategies to enhance drug permeation to improve therapeutic effects.

[0091] Example 6. Release of cell aggregates for downstream molecular analysis

[0092] The detachability of the device and the ability to extract cells from different regions greatly facilitate in-depth molecular mechanism analysis. After culture, the PDMS layer can be easily separated from the glass substrate. Cell aggregates released from the glass substrate are suitable for downstream mechanism analysis, including histological sectioning and H&E staining ( Figure 8 A and B). The specific method is: remove the PDMS layer on the glass substrate, and then fix the remaining cell aggregates on the chip with 4% paraformaldehyde. After fixation, the cell aggregates are carefully separated from the glass substrate with a thin scalpel. Subsequently, the cell aggregates are subjected to standard tissue sectioning procedures, followed by standard hematoxylin and eosin (HE) staining steps and immunofluorescence staining. Images were taken by Nikon Eclipse Ti2 inverted microscope. Testing of two EMT-related markers confirmed successful immunofluorescence staining ( Figure 8 C) Conventional tumor spheres are usually several hundred microns in diameter, making standard direct immunofluorescence staining challenging due to the slow antibody penetration during the incubation phase.

[0093] In addition, the smaller size of tumor spheres significantly reduced the success rate of slicing. The cell aggregates on the chip have a diameter of 6 mm, and longitudinal slicing can easily obtain hundreds of paraffin sections, providing strong material support for subsequent mechanism analysis ( Fig. 9 ).

[0094] Example 7: Gene expression profiles of cells in different regions

[0095] To further explore the application prospects of the chip, we used punching to segment the released cell aggregates, which can easily obtain cells from the inner, middle, and outer regions, each of which contains different gradient environment information.

[0096] Subsequently, the aggregate was divided into inner, middle and outer layers using a punch. Fig. 9 ). Cell aggregates separated from different areas were transferred to DMEM medium supplemented with collagenase (500U / mL) and then incubated at 37°C for 15-30 minutes to release the cells from Cell-GelMA. The obtained cells were washed twice with PBS, and then 1mL Trizol (T9108, Takara) was added for standard RNA extraction. After RNA extraction, reverse transcription was performed using PrimeScrip RT MasterMix (RR036, Takara) according to standard procedures, followed by qPCR analysis (QuantStudio 12K Flex).

[0097] qPCR analysis results showed that the expression of cell cycle-related genes decreased significantly from the outside to the inside, consistent with previous studies, indicating that cells are in a low activity state in the quiescent and necrotic areas ( Fig.10 ).

Claims

1. A microfluidic device, comprising a substrate and a capping layer, wherein the capping layer has a recessed area, wherein the recessed area has three layers of microcolumns arranged in a concentric circle shape, wherein the height of the microcolumns is the same as the depth of the recessed area, the distance between microcolumns in the same layer is 100 μm, and the interval between different layers is 100 μm, and the center position of the concentric circle has a perforation penetrating the capping layer; Preferably, the depth of the recessed area is 100-1000 μm, including 100 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm, 1000 μm; Most preferably, the depth of the recessed area is 300 μm.

2. The microfluidic device according to claim 1, wherein the height of the microcolumn is 300 μm; Preferably, the diameter of the largest circle among the concentric circles is 1000-10000 μm; Preferably, the diameter of the largest circle in the concentric circles includes 1000 μm, 2000 μm, 3000 μm, 4000 μm, 5000 μm, 6000 μm, 7000 μm, 8000 μm, 9000 μm, and 10000 μm; Most preferably, the diameter of the largest circle among the concentric circles is 6000 μm; Preferably, the cross section of the microcolumn has a length of 100 μm and a width of 50 μm; Preferably, one or more columns with a height of 300 μm may be designed in the area surrounded by the three layers of micro-columns arranged in a concentric circle shape; Preferably, the diameter of the perforations is 1 mm.

3. The microfluidic device according to claim 1, wherein the recessed area is of any shape; Preferably, the recessed area is circular; More preferably, the center of the concave area is located at the same position as the center of the three layers of microcolumns arranged in a concentric circle shape; More preferably, the diameter of the recessed area is adjusted according to the diameter of the largest circle in the concentric circles. When the diameter of the largest circle in the concentric circles is 6000 μm, the diameter of the recessed area is preferably 12 mm. Preferably, the cover layer has one or more holes, and the holes are connected to the recessed area; Preferably, the hole can be of any shape, preferably circular; Preferably, the diameter of the substrate is 34 mm; Preferably, the substrate is transparent; Preferably, the substrate material may be silicon, glass, or ceramic; Preferably, the substrate is made of glass; Preferably, the material of the capping layer is selected from one of polydimethylsiloxane, acrylonitrile-butadiene-styrene copolymer, polycarbonate, polymethyl methacrylate, polyurethane, polyethylene, polypropylene, polymethylpentene, polytetrafluoroethylene, polyvinyl chloride, cyclic polyolefin copolymer, polyvinylidene fluoride, polystyrene, polysulfone, nylon, styrene-acrylic acid copolymer, natural rubber, polyisoprene, butyl rubber, halogenated butyl rubber, polybutadiene, styrene-butadiene rubber, nitrile rubber, chloroprene rubber, ethylene-propylene rubber, epichlorohydrin rubber, polyacrylate rubber, silicone rubber, fluorosilicone rubber, fluorine-containing elastomer, perfluoroelastomer, ethylene-vinyl acetate, elastic protein, elastin, polyimide, and phenolic resin, or a mixture of any two or more thereof; Preferably, the material of the capping layer is selected from polydimethylsiloxane; Preferably, when in use, the capping layer is placed on the substrate, the recessed area and the substrate form a cavity (central cell chamber), and the cell-GelMA mixture containing cells is loaded through the perforation (cell loading entrance). The three-layer concentric circle structure of the capping layer arranges the microcolumns to prevent cell penetration and loss; the cell culture medium is added to the holes, the cell culture medium can penetrate into the cavity and provide the cell culture with the components required for cell growth, forming a gradient characteristic similar to that of a tumor spheroid, including gradient characteristics of pH, hypoxia and hardness.

4. Use of the microfluidic device according to claim 1 in simulating a gradient microenvironment in a tumor sphere.

5. A method for simulating a gradient microenvironment in a tumor spheroid in a 2D plane, the method comprising assembling a substrate and a capping layer of the microfluidic device of claim 1, adding a mixture of cells and GelMA into a cavity formed by the substrate and the capping layer through a perforation on the capping layer, adding a cell culture medium into the holes of the capping layer, and placing the mixture and the cell culture medium in an environment with standard cell culture conditions for cell culture after the addition of the mixture; Preferably, the mixture covers the cavity; Preferably, after the mixture is added to the cavity, the perforations are closed using a sheet.

6. The method of claim 5, wherein the number of cells in the mixture is less than 1×10 6 cells / 10μL GelMA; Preferably, the ratio of GelMA to cells in the mixture can be 1-10×10 5 cells / 10μL GelMA, including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10×10 5 cells / 10 μL GelMA; Preferably, the standard cell culture conditions are 37°C, 5% CO 2 Culture in a humidified incubator; Preferably, the microfluidic device is placed in a 6-well plate and then placed in a culture environment, and the gaps outside the wells of the 6-well plate are filled with sterile water; Preferably, the cells are cultured for 1-10 or more days.

7. The method of claim 5, wherein the cells include any cells derived from humans, gorillas, monkeys, horses, cattle, sheep, pigs, donkeys, camels, dogs, rabbits, cats, rats, mice, fish, birds or insects; Preferably, the cells include primary cells, tissue-derived stem cells, tissue-derived precursor cells, induced pluripotent stem cells, differentiated cells, transdifferentiation cells, cells obtained by cell engineering, tumor stem cells, tumor cells isolated from tumor tissue, fibroblasts, stromal cells, immune cells, cell strains, cell lines, insect cells, cell agglomerates, tissue agglomerates, and one or more of in vitro cultured organs. Preferably, the culture medium includes one or more of DMEM / F12 cell culture medium, William's E cell culture medium, Neurobasal Medium cell culture medium, MEM cell culture medium, DMEM cell culture medium, 1640RPMI cell culture medium, or F12 cell culture medium.

8. A method for preparing a cell slice having a gradient microenvironment, the method comprising a step of culturing cells and a step of fixing, The step of culturing cells is: assembling the substrate and the cover layer of the microfluidic device of claim 1, adding the mixture of cells and GelMA into the cavity formed by the substrate and the cover layer through the perforations on the cover layer, adding cell culture medium into the holes of the cover layer, and placing the mixture and the cell culture medium in an environment with standard cell culture conditions for cell culture after adding the mixture and the cell culture medium; The fixing step is: separating the substrate and the cover layer, and adding a fixing solution to the cells remaining on the substrate for fixing.

9. The method of claim 8, wherein the fixing solution comprises 4% paraformaldehyde; Preferably, the cell slices can be used for routine detection; Preferably, the conventional detection includes H&E staining, fluorescent staining, and PCR detection; Preferably, the slice is segmentable.

10. The cell slice prepared by the method of claim 8; Preferably, the cell characteristics in the cell slice are distributed in a gradient; Preferably, the cell properties include proliferation state, pH value, oxygen level and hardness characteristics.

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