Circular chamber organ chip and method for efficient culture of tumor balls

Abstract

The invention belongs to the technical field of biomedical engineering and microfluidics, and particularly discloses a circular chamber organ chip for efficient tumor sphere culture, the circular chamber organ chip comprises a runner layer and a bottom layer which are stacked, and the lower surface of the runner layer and the bottom layer are sealed through bonding; a culture solution channel is arranged on the lower surface of the runner layer, snakelike pipelines are symmetrically arranged on the culture solution channel, and a first liquid injection channel and a second liquid injection channel are arranged at the two ends of the culture solution channel respectively; a cell culture chamber communicated with the culture solution channel is arranged on the lower surface of the runner layer; according to the cell culture chamber disclosed by the invention, by adopting a circular tissue chamber structure, the aggregation behavior of cells in a three-dimensional matrix can be obviously optimized, the cell wall attachment tendency and the asymmetric wall attachment probability are obviously reduced, and a plurality of tumor cells can be promoted to quickly and spontaneously form a sphere-like structure in a chip; the probability that the tumor balls are irregular in shape and uneven in growth is reduced, the tumor balls can be conveniently cultured, and a standardized and repeatable three-dimensional tumor model can be established.

Description

Technical Field

[0001] This invention belongs to the fields of biomedical engineering and microfluidics, specifically relating to a circular chamber organ-on-a-chip and method for efficient culture of tumor spheres. Background Technology

[0002] Organ-on-a-chip is a microfluidic cell culture system manufactured using microfabrication technology. It can reproduce the physiological and pathological characteristics of organs in vivo by constructing in vitro models. Organ-on-a-chip can not only simulate the functions of multiple organs, but also achieve a high degree of control over cell-cell, cell-matrix and fluid environments. It has shown great potential in disease modeling, drug screening and toxicology testing, and has become an important tool for accelerating new drug development and mechanism research.

[0003] Due to their high heterogeneity and complex microenvironment, tumors place higher demands on the physiological reproduction capabilities of models. In particular, the construction of high-fidelity in vitro tumor models has become a key focus and challenge in the screening of anticancer drugs and the study of tumorigenesis mechanisms. Although traditional two-dimensional cell culture methods are widely used in drug testing, they cannot realistically simulate the three-dimensional microenvironment of tumors in vivo, resulting in significant differences between experimental results and clinical manifestations.

[0004] Three-dimensional tumor sphere culture technology has become an important means of constructing in vitro tumor models due to its good simulation of tumor tissue in terms of spatial structure, diffusion gradient and cell interaction. However, traditional three-dimensional culture methods mostly use static systems, such as low-adhesion culture dishes or ultra-low adsorption U-shaped plates, which make it difficult to achieve dynamic control of cell mechanical environment, nutrient supply and metabolic waste removal, which is not conducive to the research needs of long cycle and strong controllability.

[0005] Microfluidic organ-on-a-chip platforms not only have the ability to precisely control the fluid microenvironment, but also can realize the spatial and temporal regulation of the cellular microenvironment, making them an ideal carrier for dynamically culturing three-dimensional tumor models. Currently, the tissue chambers of mainstream tumor chips mostly adopt rectangular or linear configurations. These geometric structures are prone to asymmetric adhesion in the early stage of cell aggregation, resulting in irregular tumor sphere morphology and uneven growth, which in turn affects the stability of the model and the reproducibility of experiments. Summary of the Invention

[0006] To address the problems of asymmetric adhesion, irregular tumor sphere morphology, and uneven growth in current organ-on-a-chip systems, this invention provides a circular chamber organ-on-a-chip and method for efficient tumor sphere culture.

[0007] To achieve the above objectives, the present invention is implemented through the following technical solution: A circular chamber organ-on-a-chip for efficient tumor sphere culture includes a stacked channel layer and a bottom layer, with the lower surface of the channel layer and the bottom layer sealed by bonding; the lower surface of the channel layer has a culture medium channel, and serpentine tubes are symmetrically arranged on the culture medium channel, with a first injection channel and a second injection channel at each end of the culture medium channel; the lower surface of the channel layer has a cell culture chamber connected to the culture medium channel.

[0008] Preferably, the culture medium channel includes a first culture medium channel and a second culture medium channel, with the two ends of the first culture medium channel respectively connected to the two ends of the second culture medium channel via a third culture channel; serpentine tubes are respectively arranged on the first culture medium channel and the second culture medium channel; a first injection channel is arranged at the end of the first culture medium channel away from the second culture medium channel; and a second injection channel is arranged at the end of the second culture medium channel away from the first culture medium channel.

[0009] Preferably, the cell culture chamber is connected to the third culture channel on both sides by micropillars; the cell culture chamber is provided with a gel dispensing channel and a gel injection channel at both ends; the function of the micropillars is to form a gel-liquid interface through surface tension, blocking the cell gel but allowing small molecules in the culture medium to pass through and exchange material information.

[0010] Preferably, a capillary burst valve is provided on the lower surface of the flow channel layer, and the two ends of the capillary burst valve are respectively connected to the gel injection channel.

[0011] Preferably, the center of the capillary rupture valve is connected to the rupture valve orifice disposed on the flow channel layer.

[0012] Preferably, the flow channel layer is provided with a first injection hole communicating with the first injection channel, a second injection hole communicating with the second injection channel, a gel injection hole communicating with the gel injection channel, and a gel injection hole communicating with the gel injection channel.

[0013] Preferably, the upper surface of the flow channel layer is provided with a first liquid storage pipe connected to the first liquid injection hole and a second liquid storage pipe connected to the second liquid injection hole.

[0014] Preferably, the first injection channel and the second injection channel are rhomboid injection cavities; the cell culture chamber is a circular tissue chamber with continuous boundaries, and the cell culture chamber includes multiple circular tissue chambers, which are connected by a cross-section.

[0015] Preferably, the widths of the gel injection channel and the gel dispensing channel gradually decrease towards the cell culture chamber side along the gel injection hole and the gel dispensing hole, respectively.

[0016] Preferably, the thickness of the flow channel layer is 1-2 mm; the thickness of the culture medium channel is 100 μm; the diameter of the first injection hole is 1-1.5 mm; the diameter of the second injection hole is 1-1.5 mm; the diameter of the gel dispensing hole is 1-1.5 mm; the diameter of the gel injection hole is 1-1.5 mm; the diameter of the burst valve hole is 1-1.5 mm; the length of the serpentine pipe is 4-5 mm; the length of the culture medium channel is 10-12 mm and the width is 0.5 mm; the diameter of the glass tube is 8 mm and the height is 11 mm; and the thickness of the bottom layer is 1-2 mm.

[0017] A method for efficient culture of circular chamber organ-on-a-chip tumor spheres, comprising the following steps: S1. Draw the structure of each channel on the channel layer, make a mask, make a channel layer silicon wafer by photolithography, and cast it to form a monolithic structure. Then, by drilling holes, bonding the channel layer to the bottom layer, and attaching a glass tube to the surface of the channel layer, the organ chip is obtained.

[0018] S2. The organ-on-a-chip is sterilized at high temperature for 30 minutes and then dried for later use.

[0019] S3. After UV sterilization for 30 minutes, hydrogel containing liver cancer cells and thrombin was infused into the gel injection wells of the organ-on-a-chip. The organ-on-a-chip was then placed in a cell culture incubator for 10 minutes to allow the cells to adhere and grow, preventing them from being washed away during the injection of culture medium. The concentration of liver cancer cells in the hydrogel was 1.5 × 10⁻⁶. 5 / ml; the parameters of the incubator are CO2 content of 5% and temperature of 37℃.

[0020] S4. After the cells adhere to the cell culture medium, remove the organ-on-a-chip and inject collagen into it through the first or second injection well for modification. The collagen enters the cell culture chamber through the first or second injection channel, the culture medium channel, and the serpentine tube, and emerges from the second or first injection well. Then, place the organ-on-a-chip in an incubator for 10 minutes; the collagen content is 10 μl.

[0021] S5. Remove the organ-on-a-chip from the incubator and add EGM-2 (endothelial cell culture medium) to two glass tubes respectively. The first reservoir contains 1.2 ml of culture medium and the second reservoir contains 0.2 ml of culture medium. Then put the organ-on-a-chip back into the incubator to continue culturing and obtain experimental results.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This invention adopts a double-layer structure of a flow channel layer and a bottom layer, which has the characteristics of simple structure, convenient operation, easy chip fabrication, and good biocompatibility. The cell culture chamber of this invention adopts a circular tissue chamber configuration, which can significantly optimize the aggregation behavior of cells in the three-dimensional matrix, significantly reduce the cell adhesion tendency and the probability of asymmetric adhesion, and promote the rapid and spontaneous formation of spheroidal structures of various tumor cells in the chip, reduce the probability of irregular tumor sphere morphology and uneven growth, facilitate the culture of tumor spheres, and help to establish standardized and reproducible three-dimensional tumor models.

[0023] 2. The cell culture chamber of this invention adopts a circular tissue chamber configuration. The cell culture chamber exchanges material information with the culture medium channel through the gap between the micropillars. The micropillars can form a gel interface through surface tension, preventing the cell gel in the cell culture chamber from flowing into the culture medium channel. However, small molecules in the culture medium in the culture medium channel can enter the cell culture chamber through the micropillars and exchange material information, providing material information support for the circular tissue chamber cell culture chamber. This can reduce the impact on the cells cultured in the cell culture chamber, improve the performance and reliability of organ-on-a-chip models, and at the same time, the circular tissue chamber cell culture chamber can significantly reduce the cell adhesion tendency and the probability of asymmetric adhesion, and can promote the rapid and spontaneous formation of spheroidal structures by various tumor cells, which is convenient for culturing tumor spheres.

[0024] 3. This invention can be applied to multiple areas such as drug sensitivity testing, tumor stem cell screening, and anti-angiogenesis research. It can also be combined with upstream cell transfection, gene editing, and other operations to provide an efficient platform for tumor mechanism research and preclinical evaluation. Attached Figure Description

[0025] Figure 1 This is a schematic diagram of the exploded structure of the present invention in Example 1; Figure 2 This is a schematic diagram of the overall structure of the present invention in Embodiment 1; Figure 3 This is a perspective structural diagram of the present invention in Embodiment 1; Figure 4 This is a schematic diagram of the lower surface structure of the flow channel layer in Example 1; Figure 5 This is a schematic diagram of the experimental results in Example 3; Figure 6 This is a schematic diagram of the experimental results in Example 4; Figure 7 This is a schematic diagram of the experimental results of multi-cell co-culture in Example 5.

[0026] In the figure, glass tube 1; flow channel layer 2; bottom layer 3; first liquid storage tube 11; second liquid storage tube 12; first liquid injection channel 21; second liquid injection channel 22; serpentine tube 23; microcolumn 25; cell culture chamber 26; capillary burst valve 27; first liquid injection hole 211; second liquid injection hole 212; gel injection hole 213; burst valve hole 214; gel injection hole 215; gel injection channel 216; first culture medium channel 217; second culture medium channel 218; third culture channel 219; gel injection channel 220. Detailed Implementation

[0027] The present invention will be further described in detail below through specific embodiments, but this does not limit the scope of the present invention.

[0028] Example 1 A circular chamber organ-on-a-chip for efficient culture of tumor spheres, its structure is as follows: Figure 1-4 As shown, it includes a layered flow channel layer 2 and a bottom layer 3, with the lower surface of the flow channel layer 2 and the bottom layer 3 sealed by bonding; the lower surface of the flow channel layer 2 is provided with a culture medium channel, and serpentine pipes 23 are symmetrically arranged on the culture medium channel; the two ends of the culture medium channel are respectively provided with a first injection channel 21 and a second injection channel 22; the lower surface of the flow channel layer 2 is provided with a cell culture chamber 26 connected to the culture medium channel.

[0029] The culture medium channel includes a first culture medium channel 217 and a second culture medium channel 218. The two ends of the first culture medium channel 217 are connected to the two ends of the second culture medium channel 218 through a third culture channel 219. A serpentine tube 23 is respectively disposed on the first culture medium channel 217 and the second culture medium channel 218. A first injection channel 21 is disposed at the end of the first culture medium channel 217 away from the second culture medium channel 218. A second injection channel 22 is disposed at the end of the second culture medium channel 218 away from the first culture medium channel 217.

[0030] The cell culture chamber 26 is connected to the third culture channel 219 on both sides via micropillars 25; the cell culture chamber 26 is provided with a gel dispensing channel 216 and a gel injection channel 220 at both ends.

[0031] A capillary burst valve 27 is provided on the lower surface of the flow channel layer 2, and both ends of the capillary burst valve 27 are connected to the gel injection channel 220. The center of the capillary burst valve 27 is connected to the burst valve hole 214 provided on the flow channel layer 2.

[0032] The flow channel layer 2 is provided with a first injection hole 211 connected to the first injection channel 21, a second injection hole 212 connected to the second injection channel 22, a gel injection hole 213 connected to the gel injection channel 216, and a gel injection hole 215 connected to the gel injection channel 220. The upper surface of the flow channel layer 2 is provided with a first storage tube 11 connected to the first injection hole 211 and a second storage tube 12 connected to the second injection hole 212.

[0033] The first injection channel 21 and the second injection channel 22 are rhomboid injection cavities; the cell culture chamber 26 is a circular tissue chamber with continuous boundaries. The widths of the gel injection channel 220 and the gel dispensing channel 216 gradually decrease along the gel injection hole 215 and the gel dispensing hole 213 towards the cell culture chamber 26, respectively. S1. Using AutoCAD drawing software, the structure of each channel on the channel layer 2 is drawn, a mask is made, and a channel layer silicon wafer is made by photolithography. Then, a monolithic structure is formed by casting with PDMS (polydimethylsiloxane). The channel layer 2 is bonded to the bottom layer 3 by drilling holes for the first injection hole 211, the second injection hole 212, the gel injection hole 213, the burst valve hole 214, and the gel injection hole 215. Finally, the glass tube 1 is attached to the surface of the channel layer 2 to obtain the organ-on-a-chip.

[0034] S2. The organ-on-a-chip with cell culture chamber 26 is sterilized at high temperature for 30 minutes and then dried for later use.

[0035] S3. After UV sterilization for 30 minutes, hydrogel containing liver cancer cells and thrombin is infused into the gel injection well 215. The hydrogel flows into the cell culture chamber 26 along the gel injection channel 220, and air bubbles are expelled through the gel outlet channel 216 and gel outlet well 213, filling the cell culture chamber 26 with hydrogel. The organ-on-a-chip is then placed in a cell culture incubator for 10 minutes to allow the cells to adhere and grow, preventing them from being washed away during the injection of culture medium. The concentration of liver cancer cells in the hydrogel is 1.5 × 10⁻⁶. 5 / ml; the parameters of the incubator are CO2 content of 5% and temperature of 37℃.

[0036] S4. After the cells adhere to the cell culture chamber, the organ-on-a-chip is removed. Collagen is injected through the first injection port 211 or the second injection port 212 for modification. The collagen passes through the first injection channel 21 or the second injection channel 22, through the first culture medium channel 217 or the second culture medium channel 218 and the serpentine tube 23, and reaches the third culture channel 219 on both sides of the cell culture chamber 26. It then flows along the second culture medium channel 218 or the first culture medium channel 217, the serpentine tube 23, the second injection channel 22 or the first injection channel 21, and emerges from the second injection port 212 or the first injection port 211. The organ-on-a-chip is then placed in an incubator for 10 minutes. The collagen content is 10 μl. The capillary burst valve 27 is connected to the external atmospheric pressure through the burst valve port 214 to regulate and release the pressure when injecting the cell-containing hydrogel.

[0037] S5. Remove the organ-on-a-chip from the incubator and add EGM-2 culture medium to the two glass tubes 1 respectively. The culture medium in the first reservoir tube 11 is 1.2 ml and the culture medium in the second reservoir tube 12 is 0.2 ml. Then put the organ-on-a-chip back into the incubator to continue culturing, thereby obtaining the experimental results.

[0038] Example 2 A method for efficient culture of circular chamber organ-on-a-chip tumor spheres, comprising the following steps: The structural drawings of the flow channel layer 2 and the bottom layer 3 of the circular chamber organ-on-a-chip used for high-efficiency culture of tumor spheres in Example 1 were drawn using AutoCAD software. After being processed into a mask, a silicon wafer with a microchannel structure (the selected culture medium channel height is 200 μm) was fabricated by soft photolithography. This wafer was then attached to a culture dish with double-sided tape to form a mold. The chip configuration of the flow channel layer 2 in the mask is shown below. Figure 4 As shown; a PDMS film with microchannel structure is obtained by uniformly mixing polydimethylsiloxane (PDMS) prepolymer and curing agent in a 10:1 ratio and pouring it into a mold. Vacuuming, bubble blowing, drying, and peeling are then performed. The first injection hole 211, the second injection hole 212, the gel injection hole 213, the burst valve hole 214, and the gel injection hole 215 are then perforated. For the preparation and selection of the bottom layer 3, a PDMS film with a thickness of 2 mm is used. Specifically, the PDMS prepolymer and curing agent are uniformly mixed in a 10:1 ratio, vacuumed, poured onto the lid of a petri dish, and then spin-coated and dried.

[0039] Subsequently, the flow channel surface of the lower surface of the flow channel layer 2 and the bottom layer 3 are plasma bonded. After the bonding is completed, an organ-on-a-chip is obtained. It is placed on a hot plate and baked for 5 minutes to strengthen the bonding between the flow channel layer 2 and the bottom layer 3. Finally, a small amount of PDMS is dipped into the glass tube 1 and attached to the first injection hole 211 and the second injection hole 212 respectively. The prepared organ-on-a-chip is then placed on a hot plate for curing. The curing time is 2 hours and the curing temperature is 75°C. Finally, a circular chamber organ-on-a-chip with a liquid storage glass tube 1 for high-efficiency culture of tumor spheres is obtained. Before the experiment, this organ-on-a-chip needs to be sterilized by high temperature, drying and ultraviolet light, and stored in a clean bench.

[0040] Example 3 A method for efficient culture of circular chamber organ-on-a-chip tumor spheres, comprising the following steps: When using this organ-on-a-chip for experiments, the confluence of cells in the culture state should reach approximately 70%-80%. Trypsin is used to convert the cells from an adherent state to a floating state, and this process is terminated with a solution containing 10% FBS. After centrifugation, the supernatant is collected and added to the cell culture medium to prepare a cell density of 1×10⁻⁶ cells / mL. 6 Cell suspension of cells / ml; 150 μl of liver cancer cell suspension was added to a centrifuge tube, mixed and centrifuged, the supernatant was discarded and 100 μl of filtered hydrogel was added, and the mixture was mixed to obtain hydrogel cell mixture.

[0041] Thrombin and the obtained hydrogel cell mixture were mixed evenly at a ratio of 1:10 (within 20 seconds). An organ-on-a-chip without an injection system was selected, and hydrogel containing liver cancer cells and thrombin was perfused into the gel injection well 215. After successful injection of the cell-containing gel into the organ-on-a-chip, 10 μl of laminin was injected into the first injection well 211, and the organ-on-a-chip was placed in a cell culture incubator at 37°C and 5% carbon dioxide concentration for 10 minutes to observe whether the culture medium flow channel was smooth.

[0042] Then inject an appropriate amount of culture medium into the second injection hole 212 and observe whether the culture medium emerges from the first injection hole 211. After observing the culture medium emerge, inject 1200 μl and 200 μl of EGM-2 culture medium into the first reservoir tube 11 and the second reservoir tube 12, respectively. Under the action of gravity, the culture medium is constantly perfused into the cell channel. The culture medium provides nutrition to the cell culture chamber 26 through the microcolumn 25. The function of the microcolumn 25 is to form a gel interface through surface tension, which blocks the cell gel but allows small molecules in the culture medium to pass through and carry out material information exchange. Place the organ-on-a-chip in a cell culture incubator with a temperature of 37°C and a carbon dioxide concentration of 5% for 4 hours and observe whether there are bubbles in the culture medium channel.

[0043] If air bubbles are present, remove them using a 10 μl pipette tip, then place the cell culture incubator at 37°C and 5% CO2 concentration for further culture. The culture medium in the first reservoir 11 and the second reservoir 12 needs to be changed daily. Specifically, the culture medium in the first reservoir 11 and the second reservoir 12 should be alternately added. For example, if 200 μl of culture medium is added to the first reservoir 11 and 1200 μl to the second reservoir 12 on the first day, then 200 μl of culture medium should be added to the second reservoir 12 and 1200 μl to the first reservoir 11 on the second day. Maintaining a hydrostatic pressure difference between the culture medium in the first reservoir 11 and the second reservoir 12 continuously stimulates the cells. Experimental results are as follows: Figure 5 As shown, liver cancer cells can be observed to form spherical shapes under a microscope on the fifth day.

[0044] Example 4 A method for efficient culture of circular chamber organ-on-a-chip tumor spheres. This embodiment is otherwise identical to Embodiment 3, except that: Figure 6 As shown, a permeable vascular network (endothelial cells + fibroblasts) can be cultured in the circular chamber of this embodiment on the fifth day, and the vascular marker CD31 can be stained with immunofluorescence, indicating that the chip can generate a chip. Compared with the conventional rectangular chamber chip, the circular chamber of this embodiment has the advantage that a circle can be used as a field of view for result statistics, resulting in higher throughput.

[0045] Example 5 A method for efficient culture of tumor spheres using a circular chamber organ-on-a-chip is described in this embodiment. The other parts of this embodiment are the same as in Embodiment 3, except that the circular chamber organ-on-a-chip in this embodiment can also be used for multi-cell co-culture, such as... Figure 7 As shown, endothelial fibroblasts and HepG2 cells were mixed and injected into the chip. On the fifth day, vascular network could be observed. Immunofluorescence staining was then performed to detect the vascular marker CD31 and the HepG2 polar secretion marker multidrug resistance-associated protein 2 (MRP2).

[0046] The above description is only a preferred embodiment of the present invention, but is not limited to the above examples. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A circular chamber organ-on-a-chip for high-efficiency culture of tumor spheres, characterized in that, It includes a layered flow channel layer and a bottom layer, with the lower surface of the flow channel layer and the bottom layer sealed by bonding; the lower surface of the flow channel layer is provided with a culture medium channel, and serpentine pipes are symmetrically arranged on the culture medium channel; a first injection channel and a second injection channel are respectively provided at both ends of the culture medium channel; the lower surface of the flow channel layer is provided with a cell culture chamber connected to the culture medium channel.

2. The circular chamber organ-on-a-chip for high-efficiency culture of tumor spheres according to claim 1, characterized in that, The culture medium channel includes a first culture medium channel and a second culture medium channel. The two ends of the first culture medium channel are respectively connected to the two ends of the second culture medium channel through a third culture channel. The serpentine tubes are respectively arranged on the first culture medium channel and the second culture medium channel. The first injection channel is arranged at the end of the first culture medium channel away from the second culture medium channel. The second injection channel is arranged at the end of the second culture medium channel away from the first culture medium channel.

3. The circular chamber organ-on-a-chip for high-efficiency culture of tumor spheres according to claim 2, characterized in that, The cell culture chamber is connected to the third culture channel on both sides by microcolumns; the cell culture chamber is provided with a gel dispensing channel and a gel injection channel at both ends.

4. The circular chamber organ-on-a-chip for high-efficiency culture of tumor spheres according to claim 3, characterized in that, The lower surface of the flow channel layer is provided with a capillary burst valve, and the two ends of the capillary burst valve are respectively connected to the gel injection channel.

5. The circular chamber organ-on-a-chip for high-efficiency culture of tumor spheres according to claim 4, characterized in that, The center of the capillary burst valve is connected to the burst valve orifice disposed on the flow channel layer.

6. The circular chamber organ-on-a-chip for high-efficiency culture of tumor spheres according to claim 5, characterized in that, The flow channel layer is provided with a first injection hole connected to the first injection channel, a second injection hole connected to the second injection channel, a gel injection hole connected to the gel injection channel, and a gel injection hole connected to the gel injection channel.

7. The circular chamber organ-on-a-chip for high-efficiency culture of tumor spheres according to claim 6, characterized in that, The upper surface of the flow channel layer is provided with a first liquid storage pipe connected to the first liquid injection hole and a second liquid storage pipe connected to the second liquid injection hole.

8. The circular chamber organ-on-a-chip for high-efficiency culture of tumor spheres according to claim 7, characterized in that, The first and second injection channels are rhomboid injection cavities; the cell culture chamber is a circular tissue chamber with continuous boundaries.

9. The circular chamber organ-on-a-chip for high-efficiency culture of tumor spheres according to claim 8, characterized in that, The widths of the gel injection channel and the gel dispensing channel gradually decrease towards the cell culture chamber from the gel injection hole and the gel dispensing hole, respectively.

10. A method for efficient culture of circular chamber organ-on-a-chip tumor spheres according to any one of claims 1-9, characterized in that the steps... include: S1. Draw the structure of each channel on the channel layer, make a mask, make a channel layer silicon wafer by photolithography, and cast it to form a monolithic structure. Then, by drilling holes, bonding the channel layer to the bottom layer, and attaching a glass tube to the surface of the channel layer, the organ chip is obtained. S2. The organ-on-a-chip is sterilized at high temperature for 30 minutes and then dried for later use. S3. After the organ-on-a-chip is sterilized by ultraviolet light for 30 minutes, hydrogel containing liver cancer cells and thrombin is injected into the gel injection well. The organ-on-a-chip is then placed in a cell culture incubator for 10 minutes to allow the cells to adhere and grow, in order to prevent the cells from being washed away when the culture medium is injected. S4. After the cells adhere to the cell culture medium, remove the organ-on-a-chip and inject collagen into it through the first or second injection well for modification. The collagen enters the cell culture chamber through the first or second injection channel, the culture medium channel, and the serpentine tube, and emerges from the second or first injection well. Then, place the organ-on-a-chip in the incubator for 10 minutes. S5. Remove the organ-on-a-chip from the incubator, add EGM-2 culture medium to the two glass tubes respectively, and then put the organ-on-a-chip back into the incubator to continue culturing, thereby obtaining the experimental results.

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