A liver cancer chip based on a micropore array, its fabrication method and application

Abstract

This invention belongs to the field of liver cancer chips and drug evaluation, specifically disclosing a liver cancer chip based on a microporous array, its preparation method, and its applications. The liver cancer chip of this invention involves the overall design and modeling of a resin chip mold, including a concentration gradient generation module and a microporous array cell culture module. The resin mold is then printed using surface projection micro-stereolithography, and finally bonded using a template replication method and plasma treatment to obtain a complete liver cancer chip. The liver cancer chip prepared by this invention can be used for in vitro drug evaluation. Its concentration gradient generation module can stably generate multiple drug concentrations, while its downstream microporous array cell culture module can efficiently generate uniformly sized and highly active liver cancer cell spheroids, which is expected to be promoted and applied in personalized, high-throughput drug evaluation.

Description

Technical Field

[0001] This invention belongs to the field of liver cancer chips and drug evaluation, specifically relating to a liver cancer chip based on a micropore array and its preparation method, as well as the application of the liver cancer chip in personalized, high-throughput drug evaluation. Background Technology

[0002] Liver cancer ranks among the top three causes of cancer-related deaths worldwide, posing a significant global health challenge. As a primary malignant tumor originating from hepatocytes, liver cancer is characterized by uncontrolled cell proliferation and differentiation disorders, often accompanied by significant intratumoral and intertumoral heterogeneity. This complexity severely limits the effectiveness of traditional treatment strategies. Current treatment options include surgical resection, radiotherapy, targeted drugs, and chemotherapy, typically following standardized clinical guidelines. However, these approaches often fail to consider the molecular diversity of individual tumors, leading to poor efficacy.

[0003] Organ-on-a-chip technology provides a powerful platform for simulating key physiological and pathological functions of human tissues in vitro, and can be used to evaluate drug responses. To more accurately simulate the tumor microenvironment, researchers are gradually integrating three-dimensional (3D) tumor spheroids into microfluidic systems. These spheroids can more effectively reproduce the structural and metabolic characteristics of solid tumors, such as hypoxic environments and dense intercellular interactions. Advances in templating and scaffolding technologies have made the reproducible construction of uniform spheroids possible, thereby improving the consistency and reliability of drug sensitivity testing.

[0004] Existing organ-on-a-chip technologies still have key technological shortcomings that make it difficult to meet the needs of personalized, high-throughput clinical applications: First, the micropore array and fluid channels are conventional flush-mount designs, which allow the flowing fluid to directly impact the cells, hindering their natural sedimentation and aggregation, resulting in low efficiency of tumor cell spheroidization and poor size uniformity. Second, the biomimicry of the tumor microenvironment is insufficient. The flush-mount interconnected structure keeps the cell spheroids in a state of continuous fluid flushing, which cannot simulate the relatively closed interstitial microenvironment characteristics of tumors in vivo. It is also difficult to form the hypoxic environment and tight intercellular connections unique to solid tumors. At the same time, the activity of the cell spheroids can only be maintained for a short period of time, which cannot meet the experimental requirements for long-term drug effect evaluation. Summary of the Invention

[0005] Purpose of the invention: The technical problem to be solved by the present invention is to address the shortcomings of the prior art. The present invention provides a liver cancer chip based on a microporous array and its preparation method. By optimizing the structural design of the microporous array and fluid channels, a more realistic tumor biomimetic microenvironment is constructed, which enables efficient and uniform culture of liver cancer cell spheroids and can maintain the long-term high activity of the cell spheroids, providing a reliable technical platform for personalized and high-throughput in vitro drug evaluation of liver cancer.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] A liver cancer chip based on a microporous array is formed by tightly bonding upper and lower chip layers, and has a concentration gradient generation module composed of serpentine flow channels and a cell culture module formed by the microporous array flow channels inside.

[0008] The concentration gradient generating module includes two or more interconnected serpentine channels. Each serpentine channel includes two or more parallel serpentine channels, and the number of serpentine channels in the later stage is greater than the number of serpentine channels in the previous stage. The first stage serpentine channel has two injection channels at its front end.

[0009] The cell culture module includes a set of microporous array channels, which are arranged one-to-one at the rear end of the last stage serpentine channel; each microporous array channel has a drainage channel at its rear end.

[0010] Preferably, for liver cancer cell culture, the serpentine flow channel in the liver cancer chip of the present invention has a width of 400-500µm and a depth of 300-400µm, with a large bend radius of 500-700µm and a small bend radius of 100-200µm.

[0011] Preferably, for liver cancer cell culture, the width of the micropore array flow channel in the liver cancer chip of the present invention is 2-2.5 mm and the depth is 1-1.5 mm; the depth of a single micropore is 200-300 µm and the radius is 100-125 µm, and the center spacing between micropores is set to 125-150 µm.

[0012] The generated cell spheres are concentrated in size at 180µm to ensure that the cell spheres will not cause central necrosis due to excessive size (central necrosis is more likely to occur if the size is greater than 200µm).

[0013] Furthermore, at the end where the serpentine flow channel connects to the micropore array flow channel, a fluid buffer with a downward gradient is provided. After the fluid enters the micropore array flow channel and forms a downward gradient buffer, the drug solution in the channel will slowly flow into the micropore area at the bottom of the "reservoir". This ensures that the drug continuously and stably diffuses into the micropores and makes full contact with the cell spheres, while also counteracting the impact of the flowing liquid and maintaining the relative static state of the fluid in the micropores.

[0014] Cell seeding on the chip relies on natural sedimentation due to gravity, and the downward gradient structure provides a dedicated sedimentation space for the cells. Under the influence of gravity, they will naturally settle to lower micropore regions, significantly improving the efficiency of cell entry into the micropores.

[0015] Furthermore, the present invention also provides a method for fabricating the above-mentioned liver cancer chip based on a micropore array, comprising the following steps:

[0016] S1. Preparation of resin chip mold: First, the overall design and modeling of the resin chip mold is carried out, including the concentration gradient generation module composed of serpentine flow channels and the cell culture module composed of microporous array flow channels. Then, the resin mold is printed by surface projection micro stereolithography to obtain the upper and lower chip layer molds.

[0017] S2. Preparation of the lower layer liver cancer chip: PDMS and curing agent are mixed to obtain PDMS mixture, which is slowly poured into the lower chip layer mold until level. After vacuuming and degassing, it is cured and demolded to obtain the lower layer liver cancer chip with serpentine flow channels and microporous array flow channels.

[0018] S3. Preparation of the upper layer liver cancer chip: PDMS and curing agent are mixed to obtain PDMS mixture, which is slowly poured into the upper chip layer mold to cover it. After vacuuming and degassing, it is cured. After demolding, a uniformly thick upper layer liver cancer chip is obtained. Then, holes are drilled on the upper layer liver cancer chip to form the preset fluid inlet and outlet.

[0019] S4. Packaging of liver cancer chip: The bonding surfaces of the lower and upper liver cancer chips are cleaned and treated with plasma. Then, the bonding surfaces of the lower and upper liver cancer chips are tightly bonded together, and a strong bond is achieved by the dehydration condensation reaction of activated surface hydroxyl groups.

[0020] Preferably, in steps S2 and S3, the PDMS mixture is prepared by mixing PDMS monomer and curing agent at a mass ratio of 10:1-1.5.

[0021] Preferably, in step S4, the power of the plasma treatment is 250-350W, and the treatment time is 60-90s.

[0022] Furthermore, the present invention also claims the application of the above-described micropore array-based liver cancer chip in the culture of liver cancer cell spheres.

[0023] Furthermore, the present invention also claims the application of the above-described micropore array-based liver cancer chip in high-throughput drug evaluation.

[0024] Specifically, liver cancer cells are first cultured using a liver cancer chip, which allows liver cancer cell spheres with regular morphology and stable activity to form in the micropores of the micropore array of the liver cancer chip.

[0025] Subsequently, drugs of different concentration gradients were continuously introduced into the microporous array channel through a serpentine flow channel to detect and observe the cell viability of liver cancer cells after exposure to the drugs, thereby achieving the evaluation of different drugs and concentration gradients.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] (1) The liver cancer chip of this invention is composed of only two layers of PDMS chips bonded together, and integrates a concentration gradient generation module and a microwell array cell culture module into one unit. It is easy to prepare and has wide applications. The front end of the chip is a serpentine channel with a multi-branch structure, forming a concentration gradient generation module. When drugs and culture media are simultaneously introduced into the two infusion ports, a laminar flow can be used to form a drug concentration gradient for high-throughput drug evaluation. Compared with existing pure dynamic culture, it can be better applied to the field of personalized, high-throughput drug evaluation.

[0028] (2) Each channel module of the liver cancer chip of the present invention can efficiently generate a large number of uniformly sized cell spheroids. The uniformity of cell size affects drug penetration. The cell spheroids cultured in it still maintain good cell viability during the 5-day culture period, laying a solid foundation for subsequent drug evaluation.

[0029] (3) Compared with traditional drug evaluation, the liver cancer chip prepared by this invention can more realistically reflect the drug effect. Attached Figure Description

[0030] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0031] Figure 1 This is a flowchart illustrating the fabrication process of the liver cancer chip of this invention.

[0032] Figure 2 These are a three-dimensional view and a detailed enlarged view of the lower chip layer mold of the liver cancer chip of this invention.

[0033] Figure 3 This is a schematic diagram of a fluid buffer zone with a downward gradient between the serpentine flow channel and the microporous array flow channel in the liver cancer chip of this invention.

[0034] Figure 4 This is a physical image of the liver cancer chip prepared according to an embodiment of the present invention.

[0035] Figure 5 These are the live and dead fluorescence staining images and quantitative statistics of liver cancer cell spheroids cultured in the chip on days 1, 3, and 5 of the example.

[0036] Figure 6 This is a comparison chart of the effects of hepatocellular carcinoma microarray culture and traditional two-dimensional cell culture in drug evaluation. Detailed Implementation

[0037] The present invention can be better understood from the following embodiments.

[0038] Example 1

[0039] A liver cancer chip based on a micropore array, such as Figure 1As shown, it is composed of upper and lower chip layers tightly bonded together, with a concentration gradient generation module consisting of serpentine flow channels and a cell culture module formed by a microporous array flow channels inside.

[0040] The concentration gradient generation module includes two or more interconnected serpentine channels. Each serpentine channel includes two or more parallel serpentine channels, and the number of serpentine channels in the later stage is greater than the number of serpentine channels in the previous stage. The first-stage serpentine channel has two injection channels at its front end.

[0041] The cell culture module includes a set of microporous array channels, which are set one-to-one at the rear end of the last stage serpentine channel; each microporous array channel has a drainage channel at its rear end.

[0042] Example 2

[0043] The specific fabrication process of the liver cancer chip based on micropore array is as follows:

[0044] (1) Design and printing of resin chip molds

[0045] Since the mold is designed for PDMS fabrication of liver cancer chips, it needs to be designed using a reverse model, such as... Figure 2 As shown, the three-dimensional structure of the chip mold mainly consists of three core functional parts: a serpentine channel structure, a micropillar array structure, and a border structure. The specific design parameters of each part are as follows: the width of the serpentine channel and other related flow channels of the chip is uniformly set to 450µm, of which the large bending radius of the serpentine channel is 600µm and the small bending radius is 150µm, and the overall height of the concentration gradient module is 350µm; the height of a single micropillar in the micropillar array is 250µm and the radius is 100µm, and the center spacing between the micropillars is set to 125µm; the border structure is used to fix the overall shape of the mold, with a width of 23mm, a length of 70.2842mm, and a height of 3.8mm.

[0046] like Figure 3 As shown, the micropore array module of the liver cancer chip is designed as a "reservoir" structure with a downward gradient, which is not flush with the concentration gradient channel. The downward gradient structure of the "reservoir" will form a fluid buffer between the channel and the micropore. The drug solution in the channel will slowly flow into the micropore area at the bottom of the "reservoir", which not only ensures that the drug diffuses continuously and stably into the micropore and makes full contact with the cell sphere, but also counteracts the impact of the flowing liquid and maintains the relative static state of the fluid in the micropore.

[0047] Cell seeding on the chip relies on natural sedimentation due to gravity. The downward gradient structure of the "reservoir" provides a dedicated sedimentation space for the cells. Under the influence of gravity, they will naturally settle to lower micropore regions, significantly improving the efficiency of cell entry into the micropores. The micropore array with the reservoir structure has a depth of 1 mm.

[0048] The upper and lower chip layer molds were obtained by using a bio-3D printer to perform surface projection micro-stereolithography.

[0049] (2) Preparation of lower-layer liver cancer chip

[0050] After printing the resin chip mold, place the removed resin chip mold in a dish and soak it in an appropriate amount of anhydrous ethanol for cleaning. After cleaning, remove the mold from the anhydrous ethanol and transfer it to a UV resin cleaning and curing machine for UV irradiation for 2 hours. After UV irradiation, transfer the mold to an oven table, set the temperature to 120℃, and heat for 2 hours. Then, mix the PDMS monomer (SYLGARD 184 SILICONE ELASTOMER BASE) and curing agent (SYLGARD 184 SILICONE ELASTOMER CURING AGENT) at a mass ratio of 10:1. Transfer the mixed PDMS to a vacuum pump and perform vacuum treatment until the small air bubbles in the mixture are completely eliminated. Slowly pour the PDMS mixture into the lower chip layer mold until the PDMS mixture is flush with the mold edge. Then, place the resin mold containing the PDMS mixture back into the vacuum pump for a second vacuum treatment. After the air bubbles have completely disappeared, transfer the mold to a 70℃ constant temperature oven and heat to cure overnight. Then, the PDMS chip is gently peeled off with flat-tipped tweezers to obtain the lower-layer liver cancer chip that integrates concentration gradient channels and micropore arrays.

[0051] (3) Preparation of the upper layer liver cancer chip

[0052] Preparation of the upper layer hepatocellular carcinoma chip: PDMS monomer (SYLGARD 184 SILICONE ELASTOMER BASE) and curing agent (SYLGARD 184 SILICONE ELASTOMER CURING AGENT) were mixed evenly at a mass ratio of 10:1 and placed in a vacuum pump to eliminate air bubbles. After the air bubbles were eliminated, the PDMS mixture was slowly poured into the upper chip layer mold, allowing the PDMS to spread evenly until it completely covered the bottom of the mold, forming a film of uniform thickness. The mold was then placed in a vacuum pump again for a second vacuum treatment. After the second degassing, the mold was transferred to a 70℃ constant temperature oven for curing for 2 hours. The upper chip was then gradually peeled off from the bottom of the mold using tweezers. After peeling, using the previously prepared lower layer hepatocellular carcinoma chip as a template, the upper chip was precisely cut along the contour of the lower chip with a knife. Finally, a punch matching the size of the PE tubing was used to precisely punch holes at the preset fluid inlet and outlet positions of the top chip.

[0053] (4) Packaging of liver cancer chips

[0054] First, clean the bonding surfaces of the upper and lower chips separately using lint-free tape. After cleaning, place the top and bottom chips into a plasma processing instrument, setting the processing power to 300W and the processing time to 30-60 seconds. Immediately after plasma processing, remove both chips from the instrument. After removal, precisely press the bonding surfaces of the upper and lower chips together to ensure tight adhesion. A strong bond is achieved through the dehydration condensation reaction of activated surface hydroxyl groups, resulting in a structurally complete, well-sealed, and fully functional liver cancer organ-on-a-chip. Figure 4 As shown.

[0055] Example 3

[0056] Viability assay of cell spheroids cultured within a hepatocellular carcinoma chip based on a microporous array:

[0057] (1) Material preparation and chip pretreatment

[0058] The pre-prepared liver cancer chip, its matching PE transfer tubing, and microinfusion pump were thoroughly wiped and disinfected with 75% anhydrous ethanol, and then transferred to a laminar flow hood. 75% anhydrous ethanol was slowly poured into the chip, ensuring all fluid channels and micropore array areas were filled. Simultaneously, the built-in UV lamp of the laminar flow hood was turned on to sterilize the chip, PE tubing, microinfusion pump, and the interior of the laminar flow hood. After UV sterilization, the sterile syringe and microinfusion pump were assembled and secured according to operating procedures. One end of the sterile PE tubing was then tightly connected to the syringe outlet, and the other end was precisely aligned with the fluid inlet of the liver cancer chip, ensuring a tight connection without any loosening or leakage. Start the microinjection pump and pump PBS into the chip at a constant flow rate, repeatedly rinsing the chip channels and microwells to thoroughly remove residual ethanol. After rinsing, replace the syringe with 4% F127 solution and continue pumping this solution into the chip using the microinjection pump until the 4% F127 solution fills all areas of the chip. Incubate at room temperature in a clean bench for 2 hours. The purpose of this incubation is to modify the hydrophilic and hydrophobic properties of the chip channels and microwell walls, reducing non-specific cell adsorption on the channel walls and improving cell sedimentation efficiency into the microwells. After incubation, replace the syringe with complete culture medium and continuously rinse the chip interior for 10 minutes using the microinjection pump. During rinsing, closely observe the removal of air bubbles to ensure complete removal of small air bubbles remaining in the chip channels and microwells. After chip pretreatment, place the chip in a clean bench for later use.

[0059] (2) Cell inoculation and culture

[0060] HepG2 cell suspension was serially diluted to 5 × 10⁻⁶ cells / mL using complete culture medium containing 1% Matrigel. 6Cells were diluted to a density of 1 cell per mL, and thoroughly mixed by pipetting during dilution to ensure single-cell morphology. The cell suspension was added to a sterile syringe, fitted to a microinjection pump, and connected to the pre-treated liver cancer microarray. The cell suspension was slowly pumped into the microarray. Once the cell suspension completely filled the fluid channels, microwell array, and all functional areas of the microarray, the microinjection pump was turned off, and the microarray was placed in a clean bench for 5-10 minutes, allowing gravity to allow the cells to settle naturally into the microwells. After settling, complete culture medium containing 1% Matrigel was pumped into the microarray to slowly rinse away any remaining cells in the channels. After rinsing, the microarray was observed under a microscope to confirm successful cell seeding into the microwells. The microarray was then transferred to a 37°C, 5% CO2 incubator for further culture.

[0061] (3) Study on the life and death of cell spheroids in liver cancer microarray

[0062] On days 1, 3, and 5 of culture, the hepatocellular carcinoma microarray to be tested was removed from the incubator. The original complete culture medium containing 1% Matrigel in the syringe connected to the microinjection pump was replaced with PBS, and PBS was slowly pumped into the microarray to rinse it. After rinsing, the prepared Calcein AM / PI dual-fluorescence detection working solution was pumped in, and the microarray was transferred to an incubator for incubation in the dark for 30 minutes. Then, fluorescence observation was performed under a Nikon laser confocal microscope, and quantitative analysis and statistical analysis were performed using ImageJ image analysis software, such as... Figure 5 As shown, on day 1 of culture, the cell spheroids had just formed, and the green fluorescence was strong, indicating that the initially formed cells had good activity and no obvious apoptosis or necrosis. On day 3 of culture, the cell spheroids matured further, the green fluorescence intensity was uniform, and the number of red fluorescent spots did not increase significantly, indicating that the cell spheroids proliferated normally during culture, maintained stable activity, and did not exhibit large-scale apoptosis. On day 5 of culture, the cell spheroids maintained a regular morphology, the green fluorescence remained at a high intensity, and the red fluorescent spots remained at a very small level, without concentrated red fluorescent areas, indicating that even with long-term culture, the cell spheroids maintained good activity and no obvious apoptosis or necrosis aggregation. Statistical analysis of the cell viability using ImageJ software showed that the cell viability of the cell spheroids on days 1, 3, and 5 remained in the high-activity range of approximately 90%.

[0063] Example 4

[0064] A comparison of the application of hepatocellular carcinoma microarray technology and traditional two-dimensional cell culture technology in drug evaluation:

[0065] Simultaneously, HepG2 cells were seeded onto a liver cancer microarray and culture dishes. After overnight culture, the cells on the microarray formed cell spheroids, while the cells on the culture plate remained adherent. Subsequently, both the cell spheroids in the liver cancer microarray and the cells on the culture plate were exposed to different concentrations of three drugs (doxorubicin, sorafenib, and cisplatin) for 48 hours. Cell viability was then detected using CCK8 assay, and the cells were stained with Calcein AM / PI for observation.

[0066] Depend on Figure 6 The CCK8 results shown in the AC diagram indicate that, at the same drug equivalent concentration, three-dimensional cell spheroids consistently exhibit higher drug resistance. This complex result stems from the combined effects of multiple levels and dimensions, rooted in the simulation of key features of solid tumors in vivo by the three-dimensional structure. We also visualized the survival and death states of 3D spheroids on the chip and 2D cells in a culture dish under different concentrations of cisplatin. Figure 6 (D, E). These results demonstrate that the liver cancer chip successfully reproduced the biomimetic three-dimensional tumor microenvironment.

[0067] This invention provides a liver cancer chip based on a microporous array, its fabrication method, and its application. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A liver cancer chip based on a micropore array, characterized in that, It is made of upper and lower chip layers that are tightly bonded together, and has a concentration gradient generation module composed of serpentine flow channels and a cell culture module formed by microporous array flow channels inside; The concentration gradient generating module includes two or more interconnected serpentine channels. Each serpentine channel includes two or more parallel serpentine channels, and the number of serpentine channels in the later stage is greater than the number of serpentine channels in the previous stage. The first stage serpentine channel has two injection channels at its front end. The cell culture module includes a set of microporous array channels, which are arranged one-to-one at the rear end of the last stage serpentine channel; each microporous array channel has a drainage channel at its rear end.

2. The liver cancer chip based on a micropore array according to claim 1, characterized in that, The serpentine flow channel has a width of 400-500µm and a depth of 300-400µm. The large bend radius in the serpentine flow channel is 500-700µm and the small bend radius is 100-200µm.

3. The liver cancer chip based on a micropore array according to claim 1, characterized in that, The width of the micropore array flow channel is 2-2.5 mm and the depth is 1-1.5 mm; the depth of a single micropore is 200-300 µm and the radius is 100-125 µm, and the center spacing between micropores is set to 125-150 µm.

4. The liver cancer chip based on a micropore array according to claim 1, characterized in that, At the end where the serpentine flow channel connects to the microporous array flow channel, a fluid buffer with a downward gradient is provided.

5. The method for fabricating the liver cancer chip based on a microporous array as described in claim 1, characterized in that, Includes the following steps: S1. Preparation of resin chip mold: First, the overall design and modeling of the resin chip mold is carried out, including the concentration gradient generation module composed of serpentine flow channels and the cell culture module composed of microporous array flow channels. Then, the resin mold is printed by surface projection micro stereolithography to obtain the upper and lower chip layer molds. S2. Preparation of the lower layer liver cancer chip: PDMS and curing agent are mixed to obtain PDMS mixture, which is slowly poured into the lower chip layer mold until level. After vacuuming and degassing, it is cured and demolded to obtain the lower layer liver cancer chip with serpentine flow channels and microporous array flow channels. S3. Preparation of the upper layer liver cancer chip: PDMS and curing agent are mixed to obtain PDMS mixture, which is slowly poured into the upper chip layer mold to cover it. After vacuuming and degassing, it is cured. After demolding, a uniformly thick upper layer liver cancer chip is obtained. Then, holes are drilled on the upper layer liver cancer chip to form the preset fluid inlet and outlet. S4. Packaging of liver cancer chip: The bonding surfaces of the lower and upper liver cancer chips are cleaned and treated with plasma. Then, the bonding surfaces of the lower and upper liver cancer chips are tightly bonded together, and a strong bond is achieved by the dehydration condensation reaction of activated surface hydroxyl groups.

6. The method for fabricating a liver cancer chip based on a microporous array according to claim 5, characterized in that, In steps S2 and S3, the PDMS mixture is prepared by mixing PDMS monomer and curing agent at a mass ratio of 10:1-1.

5.

7. The method for fabricating a liver cancer chip based on a micropore array according to claim 5, characterized in that, In step S4, the plasma treatment power is 250-350W and the treatment time is 60-90s.

8. The application of the liver cancer chip based on micropore array as described in claim 1 in culturing liver cancer cell spheroids.

9. The application of the micropore array-based liver cancer chip as described in claim 1 in high-throughput drug evaluation.

10. The application according to claim 9, characterized in that, First, liver cancer cells are cultured using a liver cancer chip, which allows liver cancer cell spheres with regular morphology and stable activity to form in the micropores of the micropore array of the liver cancer chip. Subsequently, drugs of different concentration gradients were continuously introduced into the microporous array channel through a serpentine flow channel to detect and observe the cell viability of liver cancer cells after exposure to the drugs, thereby achieving the evaluation of different drugs and concentration gradients.

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