Micro-fluidic volute chip for high-throughput three-dimensional tumor sphere culture and application of micro-fluidic volute chip

Abstract

The invention discloses a microfluidic volute chip for high-throughput three-dimensional tumor sphere culture, and belongs to the technical field of organ chips. The overall structure of the chip is in a double-layer stacking design, and the upper layer is a spiral flow channel layer for controlling the flow rate of liquid; the lower layer is a culture layer, and the surface is a hemispherical micro-pit array. The chip disclosed by the invention needs a small amount of cell samples, cells can be quickly clustered, the uniformity of tumor balls is good, the size of the tumor balls meets the standard of a medicine sieve, and necrotic cores are avoided. More importantly, the overall diameter of the chip is 6 mm, the chip can be placed in a small hole of a standard 96-well plate, that is, 3168 tumor balls can be cultured at a time by combining the chip and the 96-well plate, and compared with an existing culture platform, the culture flux can be greatly improved in unit area. The method is suitable for high-throughput standardized tumor ball culture, in-situ observation analysis and recovery analysis of tumor balls, and is compatible with a high-throughput instrument to be applied to the fields of pharmacodynamic screening, toxicity evaluation and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of organ chips, and in particular relates to a microfluidic volute chip for high-throughput three-dimensional tumor sphere culture and applications thereof. Background Art

[0002] With the development of modern medicine, although great progress has been made in the understanding, diagnosis, prevention and treatment of cancer, cancer is still one of the main diseases causing death. For cancer treatment, chemotherapy is one of the most commonly used anti-cancer treatment methods in clinical practice, but the approval rate of new drug development is still less than 5%, and there is still no truly effective treatment for advanced cancer.

[0003] Compared with two-dimensional monolayer cell culture and animal testing, in vitro three-dimensional culture models have advantages in in vivo structure simulation, complexity and species differences, and ethics. For in vitro tumor models, the three-dimensional tumor sphere model is a flexible model that can simulate tumors and their microenvironment from multiple aspects, including tissue morphology, medium transmission, and function. Its flexibility is specifically reflected in the fact that tumor spheres can be used as the research subject of the drug screening process in the form of single-type or heterogeneous multicellular tumor clusters, and can also be used as a key component of microenvironment research models such as vascularization.

[0004] With the development of the micro-fabrication manufacturing industry, the combination of microfluidic chips and three-dimensional cell culture can simultaneously detect physical quantities, chemical quantities and biological quantities under the conditions of a dynamic and controllable microenvironment. By building a microfluidic platform and culturing three-dimensional tumor spheres, compared with the hanging drop method, it provides a more stable and controllable culture environment and shear force stimulation; compared with the rotating flask method, it reduces the demand for cells and culture fluid and provides more uniform tumor spheres; for the ultra-low adhesion plate culture method, dynamic fluid culture is introduced to provide a continuous supply of shear force and nutrients. Therefore, the microfluidic method of culturing tumor spheres is the simplest and most effective model for anticancer drug screening and development programs, and these characteristics allow it to be widely used in cancer research. With the deepening of research, more and more research groups are focusing on the application of three-dimensional culture and microfluidic chips in basic research, drug screening, personalized precision medicine and POCT instant diagnosis.

[0005] At present, there are still the following defects in the culture system for high-throughput three-dimensional tumor sphere culture for drug screening: First, the 96-well low-adhesion culture plate that can achieve high throughput can generate a large tumor sphere in each well under ideal conditions. The size of the tumor sphere is relatively large. Compared with drug screening, tumor spheres with a diameter of less than 500 μm and no necrotic core are generally required, and dynamic culture with continuous perfusion cannot be achieved; secondly, most microfluidic chips cannot achieve high-throughput culture. A small number of chips that achieve high-throughput tumor sphere culture need to be equipped with additional detection accessories, and the design is complex. It is considered that the production cost is relatively high and has not been truly promoted and used; more importantly, the culture process of chips and tumor spheres has not achieved automation and standardization similar to the well plate culture mode. It is necessary to reduce human operation errors and provide a convenient platform for end users. Therefore, in order to achieve high-throughput tumor sphere culture on microfluidic chips, it is crucial to develop a microfluidic chip platform with a stable culture environment, dynamic perfusion, and the ability to culture tumor spheres of uniform size and in line with drug screening size that is compatible with high-throughput detection and analysis instruments. Summary of the invention

[0006] Purpose of the invention: The technical problem to be solved by the present invention is to provide a microfluidic volute chip capable of three-dimensional tumor cell culture.

[0007] Another technical problem to be solved by the present invention is to provide a chip device for drug screening and / or cell culture.

[0008] The technical problem that the present invention is to solve is to provide a microfluidic volute chip and a chip device for drug screening and / or cell culture for use in cell culture or drug screening.

[0009] The final technical problem to be solved by the present invention is to provide a high-throughput three-dimensional tumor sphere culture method that can improve throughput and perform drug screening.

[0010] Technical solution: In order to solve the above technical problems, the present invention provides a microfluidic volute chip, which is a double-layer bonding structure, the upper layer is a spiral flow channel layer, and the lower layer is a culture layer. The spiral flow channel layer is provided with a spiral channel, and a hemispherical micro-pit array is provided on the surface of the culture layer in the spiral channel. An inlet is arranged at the center of the spiral flow channel layer, and an outlet is arranged at the edge of the spiral flow channel layer.

[0011] Preferably, the overall structure of the microfluidic volute chip used for high-throughput three-dimensional tumor sphere culture is a double-layer cylindrical structure with a diameter of 6 mm, including a flow channel layer and a culture layer stacked in sequence, the two layers are tightly fitted and sealed, and only an inlet is set at the center of the flow channel layer and an outlet is set at the edge position for liquid to flow in and out to provide nutrition or drug stimulation for the culture layer.

[0012] Wherein, the width of the spiral channel is 0.5-0.6 mm, and the height is 0.2-0.6 mm.

[0013] Wherein, the hemispherical micropit array includes at least 33 hemispherical micropits with a diameter of 400-500 μm.

[0014] Furthermore, the culture layer has 33 hemispherical micro-pit arrays with a diameter of 500 μm distributed according to the flow channel layer, which are used for the culture of tumor spheres.

[0015] Wherein, the materials of the spiral flow channel layer and the culture layer are selected from PDMS.

[0016] Wherein, the width of the spiral channel is 0.1 mm longer than the diameter of the hemispherical micro-pit.

[0017] The present invention also includes a chip device for drug screening and / or cell culture, which includes the microfluidic volute chip and a well plate adapted to the chip.

[0018] Wherein, the well plate includes a 96-well, 24-well, 12-well or 6-well plate.

[0019] The present invention also includes the use of the microfluidic volute chip or the chip device for drug screening and / or cell culture in cell culture or drug screening. Preferably, the cells include tumor cells. Preferably, the tumor cells are selected from tumor cell lines of human or mouse origin, co-culture of tumor cells of human or mouse origin and stromal cells, and primary tumor cells of patient or mouse origin.

[0020] The present invention also includes a high-throughput three-dimensional tumor sphere culture method, comprising the following steps:

[0021] (1) Cell anti-adhesion treatment: Pluronic F127 and phosphate buffered saline are used to prepare a solution with a mass volume ratio of 2% to 5% (W / V), which is added from the inlet of the chip and treated for 12 to 24 hours;

[0022] (2) Tumor cell inoculation: the collection density is 10 5 -5×10 6 cell / mL of tumor cell suspension, adding the tumor cell suspension from the inlet of the microfluidic volute chip or the drug screening chip device, flowing out from the outlet, and placing it in a 37° C. incubator for more than 24 hours;

[0023] (3) Perfusion culture: liquid reservoirs are set at the inlet and outlet, and gravity-driven perfusion culture is performed using the height difference or by a syringe pump. The culture method also includes using the microfluidic volute chip to connect multiple chips in series or in parallel to culture large-scale tumor cells and / or screen tumor therapeutic drugs; or using the chip device for drug screening and / or cell culture to culture cells and / or screen tumor therapeutic drugs.

[0024] The drug screening includes culturing tumor cells alone or co-culturing multiple tumor cells to screen the drug.

[0025] Furthermore, the microfluidic volute chip is placed in a standard 96-well plate, which can be applied to high-throughput in vitro tumor sphere culture and large-scale drug screening.

[0026] Furthermore, multiple microfluidic volute chips can be connected in series or in parallel to achieve expansion of the culture scale or screening of multiple drug combinations.

[0027] The technical principle of the present invention is as follows: when designing the volute chip, it is necessary to consider that the cultured tumor spheres meet the requirements of tumor drug screening applications and have high-throughput culture functions. First, the design of the chip, the spiral flow channel design eliminates the centrifugation step when the cells are perfused, which is conducive to the rapid sedimentation of cells into clusters. In addition, the cell suspension concentration is 10 5 -5×10 6 cell / mL and micropit size of 400-500μm, which limits the size of tumor spheres, without necrotic cores, and the tumor spheres meet the drug screening size; secondly, the overall arrangement of the micropit array is a circular dot matrix, which can arrange the most micropits per unit area to improve the throughput. At the same time, the arrangement distance of the micropit array makes all cells settle in the micropit area; in addition, the chip has dynamic culture capabilities, restores high-fidelity culture processes, and has independent chip culture units, which is conducive to the implementation of drug screening and realizes in situ or recovery analysis.

[0028] Beneficial effects: Compared with the prior art, the snail shell chip device for high-throughput three-dimensional tumor sphere culture of the present invention has the advantages of dynamic culture and high throughput, and is suitable for culturing a large number of high-fidelity uniform tumor cell lines or patient-derived tumor spheres of suitable size for drug screening; it is compatible with 96-well plate-adapted high-throughput screening detection and analysis instruments, and can be used for drug screening; it is suitable for direct analysis and detection of tumor spheres in situ, and the recovered tumor spheres are suitable for molecular expression analysis such as nucleic acids; each well of the 96-well plate is an independent chip unit, and each well has 33 replicates, so 3168 tumor spheres can be cultured at one time. These tumor spheres can come from 96 patients, or 96 items can be analyzed at one time, which can be used for screening the most suitable drug types, combinations, concentrations and stimulation times for tumor patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 It is a schematic diagram of the structure of a microfluidic volute chip for high-throughput three-dimensional tumor sphere culture;

[0030] Figure 2 This is a comparison of the tumor sphere culture per unit area between the snail shell chip and the commercial low-adhesion 96-well plate;

[0031] Figure 3 This is a characterization image of three-dimensional cultured tumor spheroids on a microfluidic volute chip for high-throughput three-dimensional tumor spheroid culture;

[0032] Figure 4 This is a diagram of analyzing cell activity using an ELISA reader on a microfluidic snail shell chip for high-throughput three-dimensional tumor sphere culture.

[0033] Figure 5 This is a graph showing the toxic effect of 7 μM sorafenib on liver tumor spheres. DETAILED DESCRIPTION

[0034] The present invention is further described below in conjunction with the accompanying drawings and specific implementation cases. The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

[0035] Example 1

[0036] The microfluidic volute chip for high-throughput three-dimensional tumor sphere culture of the present invention is a double-layer bonded structure, the upper layer is a spiral flow channel layer, the lower layer is a culture layer, the spiral flow channel layer is provided with a spiral channel, and a hemispherical micro-pit array is provided on the surface of the culture layer in the spiral channel. The inlet is arranged at the center position of the spiral flow channel layer, and the outlet is arranged at the edge position of the spiral flow channel layer. The width of the spiral channel is 0.6mm and the height is 0.2mm. The hemispherical micro-pit array includes 33 hemispherical micro-pits with a diameter of 500μm. The mold is made by soft lithography, laser cutting or 3D printing, and the channel layer and culture layer chips are obtained by PDMS casting. The channel layer is punched at a specified position, and then the channel layer and the culture layer are sealed by oxygen plasma treatment to implement sealing.

[0037] The present invention discloses a chip device for drug screening and / or cell culture, which comprises a microfluidic volute chip and a well plate adapted to the chip. The chip is adapted to a 96-well plate, and 96 volute chip units can be cultured simultaneously, that is, 3168 tumor spheres can be cultured at one time, thereby achieving the purpose of high-throughput dynamic culture. The tumor spheres cultured at the same time have a uniform size and meet the drug screening size, and are compatible with a high-throughput detection instrument adapted to a 96-well plate to realize real-time monitoring and recovery terminal analysis, thereby improving throughput, reducing human operation errors, and achieving the purpose of drug screening.

[0038] As shown in the figure, Figure 1 (A, B) are the overall schematic diagram and physical picture of the chip. Figure 1 (C) is a schematic diagram of the volute chip unit and each layer. Figure 1 (D, E, F) are the actual pictures of the volute chip unit and the images of each layer observed under a microscope.

[0039] Example 2 The device prepared in Example 1 is used for the cultivation of tumor cells

[0040] 1. Prepare 50 μL of a 5% (W / V) solution of Pluronic F127 (Sigma-Aldrich P2443) and phosphate buffered saline (Biosharp BL302A) as a cell anti-adhesion treatment solution and add it to the chip inlet of the microfluidic volute chip for high-throughput three-dimensional tumor sphere culture or the chip device for drug screening and / or cell culture. Replace the fresh solution every 12 hours for 24 hours. After rinsing, collect the cells at a density of 10 6 cell / mL of tumor cells, 50 μL of cell suspension was added from the chip inlet, flowed out from the outlet, and placed in a 37°C incubator for 24 hours. The inlet and outlet were then used to supplement the tumor spheres with fresh culture medium or other treatments.

[0041] 2. The cells seeded in the chip are tumor cells, which may or may not contain tumor matrix cells such as fibroblasts and vascular endothelial cells. The tumor cell supply source can be tumor cells from any mammal, such as human or mouse tumor cell lines, human or mouse tumor cells and matrix cells co-cultured, and primary tumor cells from patients or mice.

[0042] The microfluidic volute chip used for high-throughput three-dimensional tumor sphere culture is used for in vitro three-dimensional culture of tumor spheres to simulate tumor structure. It is compatible with high-throughput instruments and is used in the fields of drug efficacy screening, toxicity evaluation, etc.

[0043] Example 3 Study on the Growth of Tumor Spheroids on Chip

[0044] like Figure 1 As shown, the double-layer structural material of the chip is PDMS, which is divided into an upper flow channel layer and a lower culture layer. The overall diameter is 6 mm and can be placed in the small holes of a standard 96-well plate. Figure 1 (E) shows that the hemispherical pit has a diameter of 500 μm and a depth of 250 μm. Figure 1 The height of the channel shown in (F) is 200 μm.

[0045] Pluronic F127 (Sigma-Aldrich P2443) and phosphate buffered saline (Biosharp BL302A) were prepared with a mass volume ratio of 5% (W / V) and 50 μL of the solution was added from the chip inlet as the cell anti-adhesion treatment solution. Fresh solution was replaced every 12 hours for 24 hours. Before adding the cell suspension, 50 μL of phosphate buffered saline was added from the inlet to replace the cell anti-adhesion treatment solution, and the washing was repeated 3 times. The density of 10 6 cell / mL of human colon adenocarcinoma cells (Caco-2 cells) (ATCC cell bank) and human hepatoma cells (Hepg2 cells) (ATCC cell bank), 50 μL of cell suspension was added from the inlet of the chip, flowed out from the outlet, and placed in a 37°C incubator for 24 hours. The inlet and outlet were then used to supplement the tumor spheres with fresh culture medium or other treatments.

[0046] In this example, human colon adenocarcinoma cells (Caco-2 cells) and human liver cancer cells (Hepg2 cells) were respectively cultured at 10 6 50 μL of cell suspension was inoculated at a density of cell / mL and cultured in the chip. After liver cancer cells (Hepg2 cells) were inoculated, tumor spheres were formed in the micropits, and the growth of the tumor spheres was analyzed. Figure 2 (A) and (B) are Hepg2 cell tumor spheres grown in the snail shell chip and cultured in a 96-well low adhesion plate, respectively. The effective culture area is one well of the 96-well culture plate. It is observed that more tumor spheres of appropriate sizes can be cultured in the chip, and the tumor spheres are more compact in shape.

[0047] Figure 3 (A) Recording of growth changes and image normalization of Hepg2 cell tumor spheres cultured for ten days. Figure 3 (B) Complete image of Hepg2 cell tumor spheres cultured in the snail shell chip. Figure 3 (C) is the growth curve of Hepg2 cells (n=5). Figure 3 (D) Calcein-AM / PI live / dead staining of Hepg2 cell tumor spheres after 10 days of culture, showing that the growth of liver tumor spheres was relatively uniform, without necrotic cores, and the activity of liver tumor spheres reached more than 85%. Figure 3 (E) is a microscopic image of tumor spheres of colon adenocarcinoma Caco-2 cells cultured for 6-10 days. The above results show that a variety of tumor cells can be cultured into tumor spheres of uniform size and good activity on the chip.

[0048] Example 4 Proliferation analysis of heterogeneous multicellular tumor spheres with different cell combinations

[0049] In this example, heterogeneous multicellular tumor spheres with different cell combinations were cultured according to the method of Example 2, and the proliferation of heterogeneous multicellular tumor spheres with different cell combinations was analyzed using the Cell Counting Kit-3D kit (Biosharp C0049S). The specific steps are as follows: Pluronic F127 (Sigma-Aldrich P2443) and phosphate buffered saline (Biosharp BL302A) are prepared into a solution with a mass volume ratio of 5% (W / V) as a cell anti-adhesion treatment solution. 50 μL is added from the inlet of the chip, and the fresh solution is replaced every 12 hours for 24 hours. Before adding the cell suspension, 50 μL of phosphate buffered saline is added from the inlet to replace the cell anti-adhesion treatment solution, and the rinse is repeated 3 times. While maintaining the total number of cells at 3.33×10 5 Under the condition of 1:1, only Hepg2 cells (ATCC cell bank), HUVEC (ATCC cell bank): Hepg2 (2:1), HUVEC: Hepg2 (1:2), LX-2 (ATCC cell bank): Hepg2 (2:1), LX-2: Hepg2 (1:2), HUVEC: LX-2: Hepg2 (1:1:1) were uniformly mixed to make 50 μL cell suspension, added from the inlet of the chip, flowed out from the outlet, and cultured in a 37°C incubator. After 6 days, the chip flow channel layer was removed to reduce the effect of the flow channel layer on the absorbance value, 100 μL cell culture fluid was added, and 10 μL CCK-3D solution (Biyuntian C0049S) was added to each chip. At the same time, a well containing only a chip culture layer, a corresponding amount of cell culture fluid and CCK-3D solution but no cells were added was set as a blank control. After incubation at 37°C for 30 min, the 96-well plate was gently tapped and the absorbance value was measured at about 450 nm using an enzyme reader. Figure 4 The results show that the proliferation of heterogeneous multicellular tumor spheres with different cell combinations was analyzed using the Cell Counting Kit-3D kit (Biyuntian C0049S). Figure 4 The absorbance value results reflect that compared with tumor cell clusters of a single cell type, heterogeneous multicellular tumor spheres have higher activity and stronger proliferation ability, and the adaptability of the reaction chip to direct detection by high-throughput instruments.

[0050] Example 5 Study on drug screening of tumor cells on a chip

[0051] Drug screening experiments were performed on tumor spheres. First, the IC of sorafenib was determined under three-dimensional culture conditions. 50The value was determined. In this example, the method of Example 2 was used for culture. A solution of Pluronic F127 (Sigma-Aldrich P2443) and phosphate buffered saline (Biosharp BL302A) with a mass volume ratio of 5% (W / V) was added to the inlet of the chip at 50 μL. The fresh solution was replaced every 12 hours for 24 hours. 50 μL of phosphate buffered saline was added from the inlet to replace the cell anti-adhesion treatment solution, and the washing was repeated 3 times. Hepg2 cells were cultured at 10 6 cell / mL, add 50μL from the chip entrance, replace fresh culture medium every 12h, and add 50μL of sorafenib (0, 4, 8, 12, 16, 24, 48μM) after three days of stable culture. Replace the drug every 12h and treat for 3 days. When measuring the results, remove the chip flow channel layer to reduce the influence of the flow channel layer on the absorbance value, add 100μL of cell culture fluid, and then add 10μL of CCK-3D solution to each chip. At the same time, set up a well containing only the chip culture layer, with the corresponding amount of cell culture fluid and drug solution but no cells added as a blank control. After incubation in a 37°C incubator for 30min, gently tap the 96-well plate and use an enzyme reader to measure the absorbance value at about 450nm. According to the absorbance value obtained, statistical analysis is performed to obtain the IC 50 The value is 14.17μM ( Figure 5 B) and IC measured in a traditional 2D 96-well plate 50 Value 7.9μM Figure 5 C) In comparison, it can be seen that the three-dimensional tumor spheres are more resistant to drugs, which is consistent with the actual clinical drug use results and reports in the literature.

[0052] After getting IC 50 After the value is smaller than IC 50 The cell clusters cultured in the chip were treated with drugs at the drug concentrations of 10×10-10 and 10×10-10. 5 Under the condition of Hepg2 cells only, HUVEC:Hepg2 (2:1), LX-2:Hepg2 (2:1) were uniformly mixed to make 50 μL cell suspension, added from the inlet of the chip, flowed out from the outlet, and cultured in a 37°C incubator. Fresh culture medium was replaced every 12 hours. After stable culture for 3 days, the growth of liver tumor spheres cultured alone and co-cultured liver tumor spheres treated with 7 μM sorafenib was compared with that of the control group ( Figure 5 A). Growth is measured by measuring the diameter of the spheres. Two orthogonal diameters a and b are measured and the geometric mean diameter is calculated using ImageJ software. The mean radius (r) of the spheres is determined by the following formula:

[0053] r = 1 / 2 (a*b)1 / 2 .

[0054] According to the diameter of the cell cluster, the relative diameter was obtained with the control group as the standard, and statistical plotting was performed ( Figure 5 D). Sorafenib is a commonly used anti-liver cancer drug in clinical practice. It can be seen that after treatment, the tumor spheres are reduced and have an inhibitory effect on the growth of tumor spheres, but the inhibitory effect on tumor spheres co-cultured with liver cancer and hepatic stellate cells is weaker, which may be due to hepatic stellate cells promoting the drug resistance of liver cancer cells, which is consistent with reports in the literature. The above results show the functionality of the chip for drug screening.

Claims

1. A microfluidic volute chip, characterized in that: The microfluidic volute chip is a double-layer bonded structure, the upper layer is a spiral flow channel layer, and the lower layer is a culture layer. The spiral flow channel layer is provided with a spiral channel, and a hemispherical micro-pit array is provided on the surface of the culture layer in the spiral channel. An inlet is arranged at the center position of the spiral flow channel layer, and an outlet is arranged at the edge position of the spiral flow channel layer.

2. The microfluidic volute chip according to claim 1, characterized in that: The width of the spiral channel is 0.5-0.6 mm, and the height is 0.2-0.6 mm.

3. The microfluidic volute chip according to claim 1, characterized in that: The hemispherical micropit array includes at least 33 hemispherical micropits with a diameter of 400-500 μm.

4. The microfluidic volute chip according to claim 1, characterized in that: The materials of the spiral channel layer and the culture layer are selected from PDMS. Preferably, the width of the spiral channel is 0.05-0.15 mm longer than the diameter of the hemispherical micro-pits.

5. A chip device for drug screening and / or cell culture, characterized in that: It comprises the microfluidic volute chip as described in any one of claims 1 to 4 and a well plate adapted to the chip.

6. The chip device for drug screening and / or cell culture according to claim 5, characterized in that: The well plates include 96-, 24-, 12- or 6-well plates.

7. Use of the microfluidic volute chip according to any one of claims 1 to 4 or the chip device for drug screening and / or cell culture according to claim 5 or 6 in cell culture or drug screening, preferably, the cells include tumor cells, preferably, the tumor cells are selected from tumor cell lines of human or mouse origin, co-culture of tumor cells of human or mouse origin and stromal cells, and primary tumor cells of human or mouse origin.

8. A high-throughput three-dimensional tumor sphere culture method, characterized in that: The following steps are involved: (1) Cell anti-adhesion treatment: prepare a solution with a mass volume ratio of 2%-5% (W / V) using Pluronic F127 and phosphate buffered saline, add it to the inlet of the microfluidic volute chip according to any one of claims 1 to 4 or the chip device for drug screening and / or cell culture according to claim 6 or 7, and treat for 12-24 hours; (2) Tumor cell inoculation: the collection density is 10 5 -5×10 6 cell / mL of tumor cell suspension, adding the tumor cell suspension from the inlet of the microfluidic volute chip according to any one of claims 1 to 4 or the chip device for drug screening and / or cell culture according to claim 6 or 7, and flowing out from the outlet, and placing it in a 37°C incubator for more than 24 hours; (3) Perfusion culture: Set up liquid reservoirs at the inlet and outlet, and use the height difference to drive the perfusion culture by gravity or through a syringe pump.

9. The high-throughput three-dimensional tumor sphere culture method according to claim 8, characterized in that: The culture method also includes using the microfluidic volute chip described in any one of claims 1 to 4 to connect multiple chips in series or in parallel to culture large-scale tumor cells and / or screen tumor therapeutic drugs; or using the drug screening and / or cell culture chip device described in claim 6 or 7 to culture cells and / or screen tumor therapeutic drugs.

10. The high-throughput three-dimensional tumor sphere culture method according to claim 9, characterized in that: The drug screening includes culturing tumor cells alone or co-culturing multiple tumor cells to screen the drug.

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