Microfluidic device and applications thereof

Abstract

The present application relates to the field of basic medicine, and in particular to a microfluidic device and application thereof. Specifically, the present application provides a microfluidic device characterized by three annular microcolumn arrays, which can effectively project three-dimensional tumor spheroid features onto a two-dimensional plane without the need for complex tubing systems. The device helps to form a microenvironment with different layers: a peripheral proliferative layer, a middle quiescent layer, and an inner necrotic core. A continuous pH, oxygen, and hardness gradient can be effectively established by the microfluidic device of the present application, replicating the classic gradient layer distribution observed in tumor spheres.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of basic medicine, and particularly relates to a microfluidic device and application thereof. BACKGROUND

[0002] Compared with traditional two-dimensional (2D) culture systems, three-dimensional (3D) tumor spheroids can better reflect the physiological characteristics and complexity of tumors in vivo. Tumor spheroids contain basic spatial gradient distribution characteristics, which is one of the basic signs of solid tumors. This spatial characteristic is formed by the complex interaction between vascular permeability and the strong proliferative capacity of tumor cells, resulting in a gradient environment from the proximal to the distal region 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 drug resistance acquisition. Similarly, as the spheroid grows, the distance from the external microenvironment increases, leading to the establishment of chemical gradients (pH, oxygen, glucose, ATP distribution, lactic acid accumulation, etc.), thereby hindering the metabolic activity of tumor cells in the internal space. At the same time, different regions with different proliferative capacities appear, dividing the tumor spheroid into a peripheral proliferating layer, an intermediate quiescent layer and an inner necrotic core. Thanks to the significant similarity between tumor spheroids and solid tumors, the research progress based on tumor spheroid models has greatly enhanced our understanding of tumor biology and contributed to therapeutic progress.

[0003] However, traditional tumor spheroid models exhibit two inherent shortcomings, size and shape. Studies have shown that as tumor spheroids grow, the solid stress caused by cell growth and proliferation exerts a limit on their growth, with an upper limit of diameter estimated to be about 1000 μm. At this scale, downstream analysis (such as paraffin embedding and section preparation) becomes challenging, especially the extraction of cells from different proliferative regions for molecular mechanism analysis becomes extremely difficult. The second challenge is the spherical structure of tumor spheroids, in addition to using optical microscopy for optical sectioning observation, traditional inverted fluorescence microscopes are difficult to obtain, especially real-time monitoring of the internal characteristics of tumor spheroids.

[0004] Advances in microfluidic technology have addressed several challenges faced by traditional tumor spheroid models. Different microfluidic devices have been reported to develop, mainly using flow-based or diffusion-based strategies to mimic and control the gradient properties (e.g. oxygen, pH, cell viability, etc.) of tumor spheroids or solid tumors. Moreover, most of the microfluidic platforms employ transparent substrates, which simplify real-time observation of different microenvironmental zones. However, no device has been reported to establish a spatial distribution of gradient characteristics similar to tumor spheroids, especially to achieve a radial distribution from the peripheral layer to the core, and subsequently allow the extraction of cellular components from different zones for downstream molecular mechanism analysis, such as histological staining and qPCR analysis or sequencing. SUMMARY

[0005] To solve the above technical problems, we developed a novel microfluidic device (hereinafter also referred to as device or chip in the examples) that can effectively project three-dimensional tumor spheroid characteristics onto a two-dimensional plane without the need for complex plumbing systems. The device helps to form a spherical microenvironment with different layers: a peripheral proliferating layer, an intermediate quiescent layer, and an inner necrotic core. Through the microfluidic device of the present application, continuous pH, oxygen, and stiffness gradients can be effectively established, replicating the classic gradient layer distribution observed in tumor spheroids.

[0006] The device features three annular micro-pillar arrays designed to confine a mixture of cells and gelatin methacrylate (GelMA) in the central region. This design replicates the conditions required to form a tumor spheroidal microenvironment, allowing nutrients and oxygen to diffuse from the periphery to the 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, allowing easy retrieval of cells from different gradient zones for downstream molecular analysis. Compared to existing methods, the microfluidic device of the present application is more friendly and comprehensive in replicating the tumor spheroid microenvironment, with good scalability. It can serve as a versatile tool for studying gradient environments related to tumor biology research, including chemotactic effects, cell-cell interactions, drug resistance mechanisms, and immune infiltration.

[0008] In particular, the present application provides the following technical solutions:

[0009] In a first aspect, the present application provides a microfluidic device, the device having a substrate and a cover layer, the cover layer having a recessed area, the recessed area having three layers of micro-pillars arranged in a concentric circular shape, the height of the micro-pillars 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 the micro-pillars in the same layer being 100 μm, the distance between the layers being 100 μm, the center of the concentric circles having a through-hole penetrating the cover 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, 1000 μm.

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

[0012] 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, 10000 μm.

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

[0014] Preferably, the length of the cross-section of the micro-pillar is 100 μm, and the width is 50 μm. Wherein, the length is perpendicular to the side length of the radius, and the width is parallel to the side length of the radius.

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

[0016] Preferably, the diameter of the through-hole is 1 mm.

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

[0018] Preferably, the recessed area is circular.

[0019] More preferably, the center of the recessed area is the same as the center of the three layers of micro-pillars arranged in a concentric circular shape.

[0020] More preferably, the diameter of the recessed area is adjusted according to the diameter of the largest circle in the concentric circles, and when the diameter of the largest circle in 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, which are connected to the recessed area.

[0022] Preferably, the holes can be of any shape, preferably circular (approximately circular, the area overlapping with the recessed area forms a notch).

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

[0024] Preferably, the diameter of the substrate is 34 mm, so that it can be placed in a conventional 6-well plate compatible adapter for batch operation.

[0025] Preferably, the substrate is transparent.

[0026] Preferably, the substrate material can be silicon, glass, ceramic.

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

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

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

[0030] Specifically, in use, the cover layer is placed on the substrate, the recessed area forms a cavity (central cell chamber) with the substrate, the cell-GelMA containing cells is loaded through the perforation (cell loading inlet), and the micropillars arranged in the three concentric circles of the cover layer can prevent the cells from leaking; cell culture medium is added in the holes, the cell culture medium can penetrate into the cavity and provide the necessary ingredients for cell culture, forming a gradient characteristic similar to tumor spheres, including pH value, hypoxia and hardness gradient characteristics.

[0031] In another aspect, the present application provides use of the above microfluidic device in simulating gradient microenvironment in tumor spheroid.

[0032] In another aspect, the present application provides a method for simulating gradient microenvironment in 2D planar tumor spheroid, the method comprising 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 perforation on the cover layer, adding cell culture medium into the hole of the cover layer, placing in standard cell culture conditions after adding the mixture and the cell culture medium, and replacing the medium at regular intervals.

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

[0034] Preferably, after the mixture is added into the cavity, the perforation is covered (sealed) using a wafer. Specifically, a 9 mm cover glass can be used.

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

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

[0037] Preferably, the standard cell culture conditions are specifically, for example, incubation in a humidified incubator at 37℃, 5% CO2.

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

[0039] Preferably, the frequency of medium replacement and the total incubation time can be determined according to the characteristics and growth state of the cells, specifically, for example, medium replacement every two days, and a total incubation time of 1-10 or more days.

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

[0041] Preferably, the cells include one or more of primary cells, tissue-derived stem cells, tissue-derived precursor cells, induced pluripotent stem cells, differentiation-derived cells, transdifferentiation-derived cells, cell engineering-derived cells, tumor stem cells, tumor tissue-isolated tumor cells, tumor-associated fibroblasts, stromal cells, immune cells, cell strains, cell lines, insect cells, cell clumps, tissue clumps, in-vitro-cultured organs

[0042] Preferably, the culture medium can be any culture medium suitable for the target cells. For example, 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, 1640 RPMI cell culture medium, or F12 cell culture medium, etc.

[0043] In another aspect, the present application provides a method for preparing a cell slice with gradient microenvironment, the method comprising a cell culturing step and a fixing step,

[0044] The cell culturing 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, and adding cell culture medium into the holes of the cover layer. After adding the mixture and the cell culture medium, place it in standard cell culture conditions, and replace the medium at regular intervals.

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

[0046] Preferably, the slice can be divided, so that the cells in a specific area can be observed and detected.

[0047] Preferably, the fixing solution includes 4% paraformaldehyde.

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

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

[0050] Preferably, the cells in the cell slice have a gradient distribution of cell characteristics, including proliferation state, pH value, oxygen level, and stiffness characteristics, etc. BRIEF DESCRIPTION OF DRAWINGS

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

[0052] Figure 2 are result diagrams of two-dimensional planar observation of the gradient microenvironment: A is the gradient viability distribution of tumor cells on the chip, B is the relative diameter contrast of cell spheroids and cell aggregates of different sizes in suspension culture plates, C is the fluorescence image of traditional tumor spheroids under a general microscope, and D is the result diagram of cell fluorescence intensity distribution on the chip.

[0053] Figure 3 are fluorescence images obtained by culturing at different initial cell densities: A is a result diagram of the spheroidal gradient microenvironment formed at different initial cell densities, scale bar = 1000 pm, B is Figure 3 A is a statistical result diagram of fluorescence intensity.

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

[0055] Figure 5 are rhodamine penetration monitoring result diagrams of Chip_gel, A is a penetration process result diagram, B is a time series quantitative result diagram, and C is a penetration rate result diagram.

[0056] Figure 6 are rhodamine penetration monitoring result diagrams of Chip_day0, A is a penetration process result diagram, B is a time series quantitative result diagram, and C is a penetration rate result diagram.

[0057] Figure 7 are rhodamine penetration monitoring result diagrams of the chip of the completely established gradient microenvironment, A is a penetration process result diagram, B is a time series quantitative result diagram, and C is a penetration rate result diagram.

[0058] Figure 8 are a method schematic diagram of histological section of cell aggregates and detection result diagrams of H&E staining and immunofluorescence staining, A is a schematic diagram, B is an H&E staining result diagram, scale bars are 500 pm and 100 pm, respectively, and C is an immunofluorescence staining result diagram, scale bars are 1000 pm and 50 pm, respectively.

[0059] Figure 9is the physical diagram of the microfluidic device disassembly process of the present application.

[0060] Figure 10 is the gene expression detection result diagram of different area cells. DETAILED DESCRIPTION

[0061] The application will be further described below in conjunction with specific examples, but the protection scope of the application is not limited thereto, any skilled person in the art can make equivalent replacement or change according to the technical scheme and the inventive concept of the application within the technical range disclosed by the application, which should be covered in the protection scope of the application.

[0062] The materials, reagents and the like used in the following examples can be obtained from commercial channels unless otherwise specified.

[0063] Example 1, design and manufacture of microfluidic device (chip)

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

[0065] The specific manufacturing method is: using standard photolithography process to make mold, coating MicroChem SU-8 2150 resist on silicon wafer at a speed of 500 rpm for 10 seconds, and then rotating at a speed of 2000 rpm for 30 seconds. Then, soft baking, 250 mJ / cm 2 365 nm ultraviolet exposure and post-baking, to obtain the master of the device, and place it in a culture dish for standby. After mixing PDMS resin and curing agent at a ratio of 10:1, vacuum degassing, placing the mixed PDMS into the culture dish with the master, about 2-5 mm above the master surface, degassing and placing in a 70°C oven for 2h, and after cooling, carefully removing the PDMS layer from the master. According to the design, the part required by the device is cut out, i.e. the cover layer is obtained. Punch holes at the corresponding position of the cover layer, and then evenly coat a thin layer of freshly prepared PDMS at the bottom of the cover layer for bonding the glass substrate. Place the cover layer vertically on the glass substrate and bake at 80°C for 15 minutes to bond the cover layer and the glass substrate together. This assembly method ensures that the device is liquid-tight and also facilitates subsequent disassembly of the device.

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

[0067] The central region of the device is the cell chamber, with a diameter of 6 mm and a depth of 300 pm, and a 1 mm diameter hole for loading the cell-GelMA mixture. The central cell chamber is surrounded by three concentric arrays of microposts, with a length of 100 pm and a width of 50 pm. The microposts in each array are spaced 100 pm apart. This design aims 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 the crystal violet solution into the device, with the central cell chamber surrounded by the 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 lines and SK-Hep-1 cells transfected with GFP were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Invitrogen) supplemented with 10% fetal bovine serum (FBS, Invitrogen) and 1% penicillin-streptomycin (Invitrogen). Cells or cell-loaded chips were incubated at 37°C in a 5% CO2humidified incubator.

[0071] A pre-defined hole with a diameter of 1 mm was punched in the central region 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 referred to as a mixture of cells and gelatin methacrylate, which can also be referred to as cell-GelMA), and introduced into the chip through the pre-defined hole until they completely covered the region surrounded by the concentric posts. The cell-GelMA mixture was irradiated at 60 mW cm-2at 405 nm for 10 min to form a cell-GelMA hydrogel. -2The power density of the UV-LED was briefly turned on for 15 seconds to solidify the cell-GelMA. After solidification, the center area of the chip surface was covered with a thin cover glass with a diameter of 9 mm to seal the hole 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 gap outside the 6-well plate hole needs to be filled with sterile water to prevent excessive evaporation of the culture medium in the chip, and the culture medium is replaced every two days to maintain the best growth conditions.

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

[0073] Unlike the previous reported microfluidic platforms in which the cell loading inlet is usually located at the edge of the cell chamber, we strategically positioned the cell loading inlet in the center. This innovative design helps to establish a circular, closed and continuous gradient microenvironment. Comparing the relative size of the cell aggregates directly with tumor spheroids cultured in hanging dishes with diameters between 100 μm to 900 μm, it is shown that the device effectively solves the inherent size limitation Figure 2 B). The diameter of the cell aggregates is about 6 mm, which provides sufficient 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 planar observation of the cell profile within the internal region of the tumor spheroid by projecting the three-dimensional tumor spheroid features onto a two-dimensional plane Figure 2 C-D).

[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 relies on the natural penetration gradient of nutrients from the periphery to the interior, combined with the cell metabolic process. Therefore, the initial cell density plays a crucial role in determining the final result. In this application, we evaluated the effect of initial cell density on the establishment of spherical gradient microenvironment.

[0077] As shown in Figure 3 A, the cell-GelMA was loaded at a density of 1 x 10 6 cells / 10 μL GelMA, and obvious gradient characteristics appeared from the 4th day. With the increase of cell density 5 x 105 cells / 10 μL and 2.5 x 10 5 cells / 10 μL, the gradient feature appeared from day 6 and day 14, respectively. Chips with initial cell density lower than 2.5 x 10 5 cells / 10 μL were difficult to form gradient microenvironment within the 14-day 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 became lower than 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 x 10 6 cells occupy nearly 10 μL of GelMA, further increasing the cell density will make the cell-GelMA too viscous to perform loading operations, or the GelMA ratio is too low to make the cell-GelMA difficult to solidify into glue. As the gradient environment is established, the cells left in the central loading entrance gradually die, and the impact on the final microenvironment phenotype is minimal.

[0079] Example 4, Detection of the characteristics of the gradient distribution of the microenvironment

[0080] Wild-type SK-Hep1 cells were first digested with trypsin, washed with PBS, and then incubated with the corresponding dyes. The probe-labeled cells were washed once with PBS, loaded into the chip as described previously, and incubated in the incubator. After 8 hours of incubation, the 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. According to the supplier's recommendations, these compounds were dissolved in dimethyl sulfoxide (DMSO). The staining procedure was carried out according to the manufacturer's instructions. First, we used pHrodo Red AM to evaluate the pH distribution state. Figure 4A shows a gradient increase of fluorescence signal from the periphery to the center region, which means that the center region exhibits a stronger acidic microenvironment compared to the periphery. Due to the cell loading pattern mentioned before, the 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 the potential non-specific interference on the results, we introduce another probe BCECF AM to further confirm the pH distribution curve along the line of the chip. The fluorescence generation mechanism of BCECF AM is opposite to that of pHrodo Red AM, and the cells labeled by BCECF AM exhibit lower signal intensity in the lower pH microenvironment. The final results are consistent. BCECF AM staining confirms the gradient decrease of signal from the periphery to the center region, confirming the low pH level of the center region Figure 4 B). In addition, we observed that the non-specific fluorescence within the cell loading inlet was limited to a very small range, which had negligible effect on the surrounding signal. Next, the cells were stained with the hypoxia probe Image-iT Green, confirming that the cells in the center region were under high levels of hypoxic conditions, and gradually eased towards the outer region Figure 4 C).

[0082] In addition, the PDMS layer (capping layer) can be detached from the chip to expose the cell aggregates for characterization of the stiffness distribution curve, which is an operation that is difficult to achieve with traditional tumor spheroid models. The chip stiffness curve was analyzed with reference to the method previously reported (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.). Briefly, 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 using a Bioscope Catalyst atomic force microscope (Bruker, USA) in contact mode Figure 4 D) with a radius of 20 nanometers. Before each measurement, the deflection sensitivity and spring constant of each probe were calibrated on a glass slide. 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 experiment shows that the stiffness curve of the center region is significantly higher Figure 4(D) The hypoxic and pH characteristics within tumor spheroids have been widely reported for quite some time. Current research on tumor spheroid stiffness mainly focuses on overall stiffness. To our knowledge, due to the inherent limitations caused by their closed spherical structure, there are limited reports on the characterization of stiffness distribution within tumor spheroids. However, previous studies have reported that hypoxia can induce increased tumor stiffness by upregulating various collagen-modifying enzymes. This could explain our observed results.

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

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

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

[0087] We first evaluated the penetration rate of rhodamine on Chip_gel, where "Chip_gel" refers to a chip containing only GelMA and no cells. Figure 5 It was observed that it penetrated effortlessly into the internal area of ​​the chip within approximately 2 hours. Figure 5 (A and 5B), with an average diffusion rate of approximately 20.7 ± 7.3 μm / min. Figure 5 C).

[0088] We then plotted the permeation curve of rhodamine on Chip_day0, where "Chip_day0" represents a chip loaded with the GelMA-cell mixture before in vitro culture. Figure 6 In this case, the tumor microenvironment within the chip has not yet been established. Clearly, the penetration rate of rhodamine is significantly reduced in the presence of cells. Figure 6 A and 6B), the permeation rate is approximately 4.0 ± 2.4 μm / min. Figure 6 C).

[0089] In addition, the introduction of rhodamine into the well-established gradient microenvironment chip showed a longer penetration process even after an extended period of more than 24 hours. The external rhodamine was difficult to penetrate into the central region Figure 7 A and 7B), the overall diffusion rate was significantly reduced to 1.53 ± 0.2 pm / min Figure 7 C). At the end of the monitoring period, the penetration rate had almost reached zero.

[0090] Unlike previously reported microfluidic devices, the present application places the cell inlet in the central region, making the cell cavity edge of our chip form a continuous circular profile, thus realizing a concentric diffusion process from the uninterrupted edge to the center, effectively reproducing the penetration characteristics of substances into tumor spheres. Combined with the penetration curves of rhodamine in three different types of microenvironment chips, it can be seen that in the chip forming a gradient microenvironment, external substances are difficult to penetrate into the interior of the chip, eventually leading to the formation of a new concentration gradient feature in the interior of the chip. This penetration difference is also similar to the phenomenon that drugs are difficult to penetrate into the apoptotic region reported previously. Both results reveal the potential of our platform in real-time observation of drug penetration and exploration of strategies to enhance drug penetration to improve therapeutic effect.

[0091] Example 6, releasing cell aggregates for downstream molecular analysis

[0092] The detachability of the device and the ability to extract cells from different regions greatly facilitates in-depth molecular mechanism analysis. After incubation, the PDMS layer can be easily separated from the glass substrate. The 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, then fix the cell aggregates remaining 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, followed by standard hematoxylin and eosin (HE) staining steps and immunofluorescence staining. The pictures were taken by Nikon Eclipse Ti2 inverted microscope. The test of two EMT-related markers confirmed the successful immunofluorescence staining Figure 8 C). The diameter of traditional tumor spheres is usually around a few hundred microns, and due to the reduced penetration rate of antibodies during the incubation stage, standard direct immunofluorescence staining is challenging.

[0093] In addition, the smaller tumor sphere size significantly reduces the success rate of sectioning. The cell aggregates on this chip are 6 mm in diameter, and longitudinal sectioning can easily obtain hundreds of paraffin sections, providing strong material support for subsequent mechanism analysis Figure 9 ).

[0094] Example 7, Gene expression profiling of different zones of cells

[0095] To further explore the application prospect of the chip, we used punching to segment the released cell aggregates, and could easily obtain cells from the inner, middle and outer zones, each containing different gradient environmental information.

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

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

Claims

1. A microfluidic device, comprising a substrate and a cover layer, wherein the cover layer has a concave area, the microfluidic device has reservoirs at both ends, the concave area has three layers of micro-pillars arranged in a concentric circle shape, the height of the micro-pillars is the same as the depth of the concave area, the distance between the micro-pillars in the same layer is 100 μm, the distance between the micro-pillars in different layers is 100 μm, the center of the concentric circle has a through-hole penetrating the cover layer, and the through-hole is loaded with a cell-containing mixture cell-GelMA. The depth of the concave area is 100-1000 μm. The concave area has a plurality of pillars arranged in the area surrounded by the three layers of micro-pillars arranged in a concentric circle shape, which can support the spatial structure of the concave area. The cover layer is PDMS, the substrate is glass, the bottom of the cover layer is coated with a PDMS adhesive layer, and the cover layer is adhered to the substrate through the PDMS adhesive layer. 2.The microfluidic device of claim 1, wherein the height of the micro-pillars is 300 μm. 3.The microfluidic device of claim 2, wherein the diameter of the largest circle in the concentric circle is 1000-10000 μm. 4.The microfluidic device of claim 3, wherein the diameter of the largest circle in the concentric circle is 1000 μm, 2000 μm, 3000 μm, 4000 μm, 5000 μm, 6000 μm, 7000 μm, 8000 μm, 9000 μm, or 10000 μm. 5.The microfluidic device of claim 4, wherein the diameter of the largest circle in the concentric circle is 6000 μm. 6.The microfluidic device of claim 2, wherein the length of the cross-section of the micro-pillar is 100 μm and the width is 50 μm. 7.The microfluidic device of claim 1, wherein the diameter of the through-hole is 1 mm. 8.The microfluidic device of claim 1, wherein the concave area is circular. 9.The microfluidic device of claim 1, wherein the center of the concave area is the same as the center of the three layers of micro-pillars arranged in a concentric circle shape. 10.The microfluidic device of claim 9, wherein the diameter of the concave area is adjusted according to the diameter of the largest circle in the concentric circle, and when the diameter of the largest circle in the concentric circle is 6000 μm, the diameter of the concave area is 12 mm. 11.The microfluidic device of claim 1, wherein the cover layer has one or more holes, and the holes are connected to the concave area. 12.The microfluidic device of claim 11, wherein the holes are circular. 13.The microfluidic device of claim 1, wherein the diameter of the substrate is 34 mm.

14. The microfluidic device of claim 11, wherein the cover layer is placed on the substrate, the recessed area forms a central cell chamber with the substrate, the micro-pillars of the three concentric circle structure of the cover layer prevent the cells from leaking out, the pores are filled with cell culture medium, the cell culture medium penetrates into the central cell chamber and provides the cells with the necessary components for cell growth, and the cell culture medium forms a gradient characteristic similar to tumor spheroid, including pH, oxygen level and stiffness.

15. Use of the microfluidic device of claim 1 to simulate the gradient microenvironment in tumor spheroid.

16. A method for simulating tumor spheroid with gradient microenvironment in 2D plane, the method comprising using the microfluidic device of claim 14, assembling the substrate and the cover layer, adding a mixture of cells and GelMA into the central cell chamber formed by the substrate and the cover layer from the perforations on the cover layer, the number of cells in the mixture being greater than 2.5 x 10 5 cells per 10 μL of GelMA, less than 1 x 10 6 cells per 10 μL of GelMA, and adding cell culture medium into the holes of the cover layer, the microfluidic device after adding the mixture and the cell culture medium being placed in a 6-well plate, the 6-well plate containing the microfluidic device being placed in an environment with standard cell culture conditions for cell culture, the gap outside the well of the 6-well plate being filled with sterile water, and the environment with standard cell culture conditions being a humidified incubator at 37 °C, 5% CO2.

17. The method of claim 16, wherein the cell culture is performed for 1-10 days.

18. The method of claim 16, wherein the mixture covers the central cell chamber.

19. The method of claim 18, wherein the mixture is added to the central cell chamber and the pores are sealed with a thin film.

20. The method of claim 16, wherein the cells are any cells derived from human, chimpanzee, monkey, horse, cow, sheep, pig, donkey, camel, dog, rabbit, cat, rat, mouse, fish, bird or insect.

21. The method of claim 16, wherein the cell culture medium is 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, 1640 RPMI cell culture medium, and F12 cell culture medium.

22. A method of preparing a cell slice with gradient microenvironment, the method comprising a step of culturing cells and a step of fixation, the step of culturing cells is performed by the method of claim 16 in an environment with standard cell culture conditions; the step of fixation is performed by separating the substrate and the cover layer, and adding a fixative to the cells remaining on the substrate.

23. The method of claim 22, wherein the fixative is 4% paraformaldehyde.

24. The method of claim 22, wherein the cell slice is used for routine detection.

25. The method of claim 24, wherein the routine detection comprises H&E staining, fluorescent staining, and PCR detection.

26. The method of claim 22, wherein the cell slice is sectioned.

27. The cell slice prepared by the method of claim 22, wherein the cell properties are gradiently distributed in the cell slice.

28. The cell slice of claim 27, wherein the cell properties include proliferation state, pH, oxygen level, and stiffness characteristics.

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