Microfluidic device and method for cell experiment

Abstract

The invention discloses a microfluidic device and method for a cell experiment, the device comprises a packaging layer, a liquid flow layer and a culture layer, and the culture layer is provided with a plurality of groups of culture grooves; a plurality of groups of liquid injection grooves are formed in the bottom surface of the liquid flow layer, the liquid injection grooves are formed above the culture grooves and are communicated with the culture grooves, and a plurality of groups of middle-layer sample adding holes and middle-layer drainage holes are vertically formed in the two ends of each liquid injection groove; the packaging layer is provided with a plurality of groups of upper-layer sample adding holes and upper-layer drainage holes, and the upper-layer sample adding holes and the upper-layer drainage holes are respectively and correspondingly arranged above the middle-layer sample adding holes and the middle-layer drainage holes; the method comprises the following steps: S1, packaging the packaging layer, the liquid flow layer and the culture layer from top to bottom; s2, exhausting; s3, adding experimental cells; s4, an automatic liquid adding device is connected with the upper-layer sample adding hole and the upper-layer drainage hole; s5, the whole device is turned over, the cells can directly settle to a liquid flow layer, and the cells are rapidly obtained through perfusion; the invention provides a device and a method which can be used for culturing tumor organs and synchronously carrying out multiple groups of experiments.

Description

Technical Field

[0001] The invention relates to the technical field of experimental devices, in particular to a microfluidic device and method for cell experiments. Background Art

[0002] Microfluidics is a technology that integrates the basic operating units of sample preparation, reaction, separation, and detection in biological, chemical, and medical analysis processes onto a micrometer-scale chip to automatically complete the entire analysis process. Due to its huge potential in the fields of biology, chemistry, and medicine, it has developed into a new research field that intersects biology, chemistry, medicine, fluids, electronics, materials, and mechanics.

[0003] Tumor organoids are tumor cells (including tumor stem cells) isolated from patient tumor tissues, and then cultured in a microenvironment similar to that in vivo by adding certain cytokines and small molecules to form a micro-tumor model. The micro-tumor model has similar structural characteristics and functional properties to the source tumor tissues, and can be stably expanded in an in vitro 3D culture system. Tumor organoids involve three key points: the first is the material, the second is the culture scheme, and the third is the identification scheme.

[0004] The Chinese patent with publication number CN202211584503.9, a drug screening tumor organoid chip and a drug screening method, discloses a drug screening tumor organoid chip and a drug screening method. The method includes microfluidic chip preparation, two-phase aqueous solution preparation, single cell suspension preparation, microfluidic manipulation, single cell loaded microgel production and high-throughput anticancer drug screening of pancreatic cancer organoids. The invention uses a microfluidic chip with an integrated normally closed pneumatic pump valve as a technical platform, a single cell suspension pre-incubated with a cross-linker as the core of the chemical reaction, and a water-in-water droplet as a molding template. The monodispersed seed cells are loaded into a micro-hydrogel carrier by a one-step method to achieve efficient single-cell in situ loading, pancreatic cancer organoid construction and high-throughput drug screening. This method improves the consistency of the organoid culture environment and starting point, and also has the high-throughput characteristics of droplet microfluidics technology, realizing efficient and highly reproducible in vitro construction of organoids and anticancer drug screening, which can play a huge role in basic research and translational applications related to pancreatic cancer organoids.

[0005] The inventors of the present application have found in long-term experiments that the structure of the microfluidic device and the size and height of the culture tank have a significant impact on the culture of tumor organoids. Summary of the invention

[0006] The present invention overcomes these difficulties and provides a microfluidic device and method that can be used to culture tumor organoids and conduct multiple groups of experiments simultaneously.

[0007] To achieve this object, the present invention provides the following technical solutions:

[0008] The present invention provides a microfluidic device for cell experiments, comprising a packaging layer, a liquid flow layer and a culture layer from top to bottom, wherein the culture layer is provided with a plurality of groups of culture grooves, each group of the culture grooves comprising a plurality of adjacently arranged culture wells; the bottom surface of the liquid flow layer is provided with a plurality of groups of injection grooves, the injection grooves are arranged above the culture grooves, and the injection grooves are connected to the culture grooves, and a plurality of groups of middle-layer sample loading holes and middle-layer drainage holes are vertically arranged at both ends of the injection grooves; the packaging layer is provided with a plurality of groups of upper-layer sample loading holes and upper-layer drainage holes, the upper-layer sample loading holes and the upper-layer drainage holes are respectively and correspondingly arranged above the middle-layer sample loading holes and the middle-layer drainage holes.

[0009] Preferably, the height of the culture hole is 0.2-0.3 mm.

[0010] In the present invention, the height of the culture well is 0.2-0.3 mm, which can promote the aggregation of tumor organoids and will not settle to the bottom of the culture well. The height of the culture well is 0.2-0.3 mm, which does not affect the replacement of the drug solution or culture medium. The replacement of the drug solution or culture medium can be achieved by uninterrupted gas-liquid exchange and mechanical shear force flushing without affecting the aggregation of tumor organoids in the culture well.

[0011] Preferably, the height of the injection groove is 0.3-0.8 mm.

[0012] In the present invention, the height of the injection groove has a great influence on the gas-liquid exchange and the size of the mechanical shear force generated. The height of the injection groove of the present invention is 0.3-0.8 mm, which can just simulate the growth environment of cells in the body and promote normal cell growth.

[0013] Preferably, the pore diameter of the culture well is 0.2 mm.

[0014] Preferably, the surfaces of the culture tank and the injection groove are provided with a hydrophobic coating.

[0015] Preferably, the encapsulation layer, the liquid flow layer and the culture layer are all made of transparent materials.

[0016] Preferably, there are no less than 6 groups of culture tanks.

[0017] In the present invention, the culture tanks are not less than 6 groups, and each group has independent sample addition holes and drainage holes, and can be used for independent experiments. In particular, in drug concentration screening experiments, the drug concentration can be adjusted according to the progress of the experiment.

[0018] The second aspect of the present invention provides a method for cell experiment, comprising the following steps:

[0019] S1, encapsulating the encapsulation layer, the liquid flow layer and the culture layer from top to bottom;

[0020] S2. After sterilization, exhaust gas is discharged by vacuum filtration;

[0021] S3, adding experimental cells into multiple groups of culture tanks in the culture layer;

[0022] S4, the two ends of the automatic liquid adding device are connected to the upper sample adding hole and the upper drainage hole respectively. The liquid flow layer can realize uninterrupted gas-liquid exchange and mechanical shear force flushing through the automatic liquid adding device, providing an environment more similar to that in vivo for cell growth;

[0023] S5. Flip the entire device over so that the cells can directly settle into the liquid flow layer and be quickly obtained through perfusion.

[0024] Preferably, the automatic liquid adding device comprises a peristaltic pump.

[0025] Preferably, the cell experiment includes rapid in situ expansion of tumor organoid cells, rapid evaluation of multiple drugs and multiple treatment modes, and acquisition of cells after evaluation, and co-culture of tumor organoids with immune cells.

[0026] Preferably, the culture layer is provided with a plurality of groups of culture troughs, each group of the culture troughs includes a plurality of adjacently arranged culture holes; the bottom surface of the liquid flow layer is provided with a plurality of groups of liquid injection grooves, the liquid injection grooves are arranged above the culture troughs, and the liquid injection grooves are connected to the culture troughs, and a plurality of groups of middle-layer sample loading holes and middle-layer drainage holes are vertically arranged at both ends of the liquid injection grooves; the encapsulation layer is provided with a plurality of groups of upper-layer sample loading holes and upper-layer drainage holes, and the upper-layer sample loading holes and the upper-layer drainage holes are respectively correspondingly arranged above the middle-layer sample loading holes and the middle-layer drainage holes.

[0027] Preferably, the height of the culture hole is 0.2-0.3 mm.

[0028] Preferably, the height of the injection groove is 0.3-0.8 mm.

[0029] Preferably, the pore diameter of the culture well is 0.2 mm.

[0030] Preferably, the surfaces of the culture tank and the injection groove are provided with a hydrophobic coating.

[0031] Preferably, the encapsulation layer, the liquid flow layer and the culture layer are all made of transparent materials.

[0032] Preferably, there are no less than 6 groups of culture tanks.

[0033] Compared with the prior art, the present invention has the following beneficial effects and significant improvements:

[0034] 1. The microfluidic device for cell experiments of the present invention can simulate the normal in vivo environment through a unique structural design and adjustment of parameters such as the height of the culture well and the height of the injection groove, so as to create an optimal experimental environment for the culture and experiment of cells, especially tumor organoids;

[0035] 2. The present invention has confirmed through experiments that the microfluidic device for cell experiments of the present invention can be used for drug experiments of different concentrations, cell culture and acquisition experiments, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the technical solution of the present invention, the following briefly introduces the drawings required for use in the embodiments of the present invention.

[0037] Obviously, the drawings described below are only drawings of some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work, but these other drawings also belong to the drawings required for use in the embodiments of the present invention.

[0038] Figure 1 An exploded view of a microfluidic device for cell experiments according to Example 1 of the present invention;

[0039] Figure 2 A top view of a microfluidic device for cell experiments according to Example 1 of the present invention;

[0040] Figure 3 A side view of a microfluidic device for cell experiments according to Example 1 of the present invention;

[0041] Figure 4 for Figure 2 Enlarged view of point A in the middle;

[0042] Figure 5 A stereoscopic diagram of a microfluidic device for cell experiments according to Example 1 of the present invention;

[0043] Figure 6 This is a diagram of cell growth in the microfluidic device of Example 2 of the present invention;

[0044] Figure 7 The results of the cell drug sensitivity experiment in the microfluidic device of Example 3 of the present invention;

[0045] Figure 8 The results of the cell drug sensitivity test in the microplate of Example 3 of the present invention are as follows;

[0046] Fig. 9 The experimental results of chemotherapy drugs killing tumor organoids in the microfluidic device co-culture system of Example 5 of the present invention;

[0047] Fig.10 These are the experimental results of treating tumor organoids with a combination of chemotherapeutic drugs and immune checkpoint inhibitor drugs in the co-culture system of the microfluidic device of Example 5 of the present invention.

[0048] In the figure, 1, encapsulation layer, 2, liquid flow layer, 3, culture layer, 1.1, upper layer sample addition hole, 1.2, upper layer drainage hole, 2.1, middle layer sample addition hole, 2.2, middle layer drainage hole, 2.3, liquid injection groove, 3.1, culture tank. DETAILED DESCRIPTION

[0049] In order to make the purpose, technical solution, beneficial effects and significant improvements of the embodiments of the present invention more clear, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings provided in the embodiments of the present invention.

[0050] Obviously, all the described embodiments are only partial embodiments of the present invention, rather than all the embodiments; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative work are within the scope of protection of the present invention.

[0051] It should be noted that the terms "first", "second" and "third" (if any) in the specification and claims of the present invention and the drawings of the embodiments of the present invention are only used to distinguish different objects, rather than to describe a specific order. In addition, the term "comprising" and any variation thereof are intended to cover non-exclusive inclusions. For example, a process, method, system, product or device comprising a series of steps or units is not limited to the listed steps or units, but may optionally include steps or units that are not listed, or may optionally include other steps or units inherent to these processes, methods, products or devices.

[0052] What needs to be understood is:

[0053] In the present invention, unless otherwise clearly stipulated and limited, the terms such as "install", "connect", "fix" and the like should be understood in a broad sense. For example, it can be a fixed connection, a detachable connection, a movable connection, or an integrated connection; it can be a direct connection, an indirect connection through an intermediate medium, an invisible signal connection, or even an optical connection; it can be the internal connection of two elements or the interaction relationship between two elements, unless otherwise clearly limited.

[0054] For those skilled in the art, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0055] It should also be noted that the following specific embodiments may be combined with each other, and the same or similar concepts or processes therein may not be repeated in some embodiments.

[0056] The technical solution of the present invention is described in detail below with specific embodiments.

[0057] Example 1

[0058] like Figure 1-5 As shown, a microfluidic device for cell experiments comprises, from top to bottom, an encapsulation layer 1, a liquid flow layer 2 and a culture layer 3, wherein the culture layer 3 is provided with a plurality of culture tanks 3.1, and each culture tank 3.1 comprises a plurality of adjacent culture wells 3.2; a plurality of liquid injection grooves 2.3 are provided on the bottom surface of the liquid flow layer 2, wherein the liquid injection grooves 2.3 are arranged above the culture tanks 3.1, and the liquid injection grooves 2.3 are connected to the culture tanks 3.1, and a plurality of middle layer sample addition holes 2.1 and middle layer drainage holes 2.2 are vertically arranged at both ends of the liquid injection grooves 2.3; a plurality of upper layer sample addition holes 1.1 and upper layer drainage holes 1.2 are provided on the encapsulation layer 1, and the upper layer sample addition holes 1.1 and the upper layer drainage holes 1.2 are respectively arranged above the middle layer sample addition holes 2.1 and the middle layer drainage holes 2.2.

[0059] In this embodiment, the height of the culture hole 3.2 is 0.2 mm.

[0060] In this embodiment, the height of the injection groove 2.3 is 0.5 mm.

[0061] In this embodiment, the pore diameter of the culture hole 3.2 is 0.2 mm.

[0062] In this embodiment, the surfaces of the culture tank 3.1 and the injection groove 2.3 are provided with a hydrophobic coating.

[0063] In this embodiment, the encapsulation layer 1, the liquid flow layer 2 and the culture layer 3 are all made of transparent materials.

[0064] In this embodiment, there are no less than 6 groups of culture tanks 3.1.

[0065] The method for using the microfluidic device for cell experiment of this embodiment comprises the following steps:

[0066] S1, encapsulating the encapsulation layer 1, the liquid flow layer 2 and the culture layer 3 from top to bottom;

[0067] S2. After sterilization, exhaust gas is discharged by vacuum filtration;

[0068] S3, experimental cells are added to the multiple groups of culture tanks 3.1 of culture layer 3;

[0069] S4, the two ends of the automatic liquid adding device are respectively connected to the upper layer sample adding hole 1.1 and the upper layer drainage hole 1.2. The liquid flow layer 2 can realize uninterrupted gas-liquid exchange and mechanical shear force flushing through the automatic liquid adding device, providing an environment more similar to that in vivo for cell growth;

[0070] S5. Flip the entire device over, and the cells can directly settle to the liquid flow layer 2, and the cells can be quickly obtained by perfusion.

[0071] In this embodiment, the automatic liquid adding device includes a peristaltic pump.

[0072] In this embodiment, the cell experiment includes rapid in situ expansion of human tumor organoid cells, rapid evaluation of multiple drugs and multiple treatment modes, and acquisition of cells after evaluation.

[0073] Comparative Example 1

[0074] The comparative device comprises, from top to bottom, an encapsulation layer, a liquid flow layer and a culture layer, wherein the culture layer is provided with a plurality of culture troughs, each group of culture troughs comprising a plurality of adjacent culture wells; a plurality of liquid injection grooves are provided on the bottom surface of the liquid flow layer, the liquid injection grooves are arranged above the culture troughs, and the liquid injection grooves are connected to the culture troughs, and a plurality of middle-layer sample loading holes and middle-layer drainage holes are vertically arranged at both ends of the liquid injection grooves; a plurality of upper-layer sample loading holes and upper-layer drainage holes are provided on the encapsulation layer, and the upper-layer sample loading holes and the upper-layer drainage holes are respectively arranged above the middle-layer sample loading holes and the middle-layer drainage holes.

[0075] In this embodiment, the height of the culture well is 0.5 mm.

[0076] In this embodiment, the height of the injection groove is 1 mm.

[0077] In this embodiment, the pore diameter of the culture well is 0.5 mm.

[0078] In this embodiment, the surfaces of the culture tank and the injection groove are provided with a hydrophobic coating.

[0079] In this embodiment, the encapsulation layer, the liquid flow layer and the culture layer are all made of transparent materials.

[0080] In this embodiment, there are no less than 6 groups of culture tanks.

[0081] Example 2 Rapid in situ expansion and acquisition of human tumor organoid cells

[0082] The microfluidic device for cell experiments in Example 1 was used to amplify and obtain human tumor organoid cells. The amplification method is briefly summarized as follows:

[0083] Step 1: Encapsulate the encapsulation layer, liquid flow layer and culture layer from top to bottom;

[0084] Step 2: After sterilization, exhaust air using vacuum filtration;

[0085] Step 3: Add human tumor organoid cells into multiple groups of culture tanks in the culture layer;

[0086] Step 4: The two ends of the automatic liquid adding device are connected to the upper sample adding hole and the upper drainage hole respectively. The liquid flow layer can achieve uninterrupted gas-liquid exchange and mechanical shear force flushing through the automatic liquid adding device, providing an environment more similar to that in vivo for cell growth;

[0087] Step 5: Flip the entire device over so that the cells can settle directly into the liquid flow layer and human tumor organoid cells can be quickly obtained through perfusion.

[0088] Cell growth in microfluidic devices Figure 6 As shown, it can be seen that the cells grow in clusters and non-adherently. In summary, the microfluidic device of the present invention realizes the non-adherent culture of primary tumor cells, so that a small amount of human tumor primary cells can be rapidly amplified in the microfluidic device. In addition, since an uninterrupted liquid flow system is designed on the upper part of the microfluidic device, rapid gas-liquid exchange of the culture system can be achieved, overcoming the shortcomings of traditional chips due to narrow gap space and poor cell growth state. In addition, after the cells are cultured or treated, the microfluidic device body is flipped over, and the organoid cells can be directly settled in the middle liquid flow layer, and the target cells can be quickly obtained by perfusion, and used for subsequent experiments, thereby realizing the phenotypic screening and molecular difference analysis of the same cell sample, ensuring the smooth development of the subsequent drug resistance mechanism.

[0089] This embodiment also uses the device of comparative example 1 to expand and obtain human tumor organoid cells, and the specific experimental method is the same as above. As a result, the cells are all gathered at the bottom of the culture layer, and the cell growth state is poor.

[0090] Example 3 Multiple Drugs Multiple Treatment Modes

[0091] The microfluidic device and microplate (commercially available) for cell experiments in Example 1 were used to expand human tumor organoid cells. The expansion method is briefly summarized as follows:

[0092] Step 1: Cultivate cell lines in microplates and microfluidic devices respectively;

[0093] Step 2: Use the same drug and drug concentration to stimulate

[0094] Step 3: The microplate uses ATP or AO / PI method to detect cell activity; while the microfluidic device drug sensitivity detection platform uses fluorescence quantitative method to detect cell activity, and compare the differences between the two platforms.

[0095] The results are as follows Figure 7 and 8 As shown, Figure 7Results of cell drug sensitivity test in microfluidic device. Figure 8 The results of the cell drug sensitivity test in the microplate. It can be seen that the microfluidic device of the present invention can carry out multiple groups of experiments simultaneously, and the experimental cells grow in clusters and non-adherently. In summary, the microfluidic device of the present invention retains the sample addition holes and drainage holes. By designing a multi-channel system externally, it can realize the screening of multiple drug combinations under the same culture conditions, with a small cell demand, and can adjust the drug concentration at any time according to the cell's response to the drug, shortening the drug sensitivity test cycle and improving the efficiency of drug sensitivity testing; achieving medium- and long-term drug guidance, sequential drug use plans and continuous evaluation of efficacy.

[0096] Example 4 Drug sensitivity testing of body fluid samples

[0097] The microfluidic device for cell experiment in Example 1 was used to perform drug sensitivity detection of body fluid samples. The experimental method is briefly summarized as follows:

[0098] Step 1: After selective culture of the body fluid sample, the cell microclusters are injected into the microfluidic device.

[0099] Step 2: Use an automatic liquid adding device to achieve automatic circulation of drug administration and provision of nutrients;

[0100] Step 3: Detect the differences in cell activity in each channel under various drugs and drug concentrations.

[0101] Example 5 Co-culture of tumor organoids and immune cells

[0102] The microfluidic device and microplate (commercially available) for cell experiments in Example 1 were used to co-culture tumor organoids and immune cells, and the co-culture system was used to perform target-immune combined and chemo-immune combined drug reactivity detection.

[0103] Step 1: Cultivate organoids in microplates and microfluidic devices respectively;

[0104] Step 2: After the organoids are formed, immune cells labeled with fluorescent dyes are added to the microfluidic chip at a certain ratio of target effector cells to construct a co-culture system;

[0105] Step 3: Add targeted drugs, immune drugs, or both to the co-culture system for treatment; add PI dye 4-7 days after drug treatment to detect the activity of organoid cells.

[0106] The results are as follows Fig. 9 and 10 As shown, Fig. 9 The experimental results of chemotherapy drugs killing tumor organoids in the microfluidic device co-culture system are shown in Figure 2. Fig.10 These are the experimental results of combined treatment of tumor organoids with chemotherapeutic drugs and immune checkpoint inhibitors in a microfluidic device co-culture system. Fig. 9It was shown that immune cells can enhance the killing effect of low-dose chemotherapy drugs on tumor organoids; Fig.10 It is shown that the microfluidic device of the present invention can be used to evaluate the killing effect of immune checkpoint inhibitors combined with other drugs on tumor organoids.

[0107] In summary, the microfluidic device of the present invention can construct a tumor organoid immune microenvironment by adding immune cells, so that it can be applied to the multi-dimensional drug combination therapy efficacy evaluation, enriching the application scenarios of the microfluidic chip in guiding clinical drug selection.

[0108] In the description of the above manual:

[0109] The descriptions of the terms "this embodiment", "an embodiment of the present invention", "as shown in...", "further", "a further improved technical sub-scheme", etc., mean that the specific features, structures, materials or characteristics described in the embodiment or example are included in at least one embodiment or example of the present invention; in this specification, the schematic expressions of the above terms are not necessarily for the same embodiment or example, and the specific features, structures, materials or characteristics described may be combined or combined in a suitable manner in any one or more embodiments or examples; in addition, a person of ordinary skill in the art may combine or combine the different embodiments or examples and the features of the different embodiments or examples described in this specification without causing any contradiction.

[0110] Finally, it should be noted that:

[0111] The above embodiments are only used to illustrate the technical solutions of the present invention, but not to limit them.

[0112] Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some or all of the technical features therein, and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention. The non-essential improvements, adjustments or replacements made by those skilled in the art based on the contents of this specification are all within the scope of protection required by the present invention.

Claims

1. A microfluidic device for cell experiments, characterized in that: It includes the encapsulation layer, liquid flow layer and culture layer from top to bottom. The culture layer is provided with a plurality of groups of culture tanks, each group of the culture tanks comprising a plurality of adjacently arranged culture holes; The bottom surface of the liquid flow layer is provided with a plurality of groups of injection grooves, the injection grooves are arranged above the culture tank, and the injection grooves are connected to the culture tank, and the two ends of the injection grooves are vertically provided with a plurality of groups of middle layer sample loading holes and middle layer drainage holes; The packaging layer is provided with a plurality of groups of upper sample adding holes and upper drainage holes, and the upper sample adding holes and the upper drainage holes are respectively and correspondingly arranged above the middle sample adding holes and the middle drainage holes.

2. A microfluidic device for cell experiments as claimed in claim 1, characterized in that: The height of the culture hole is 0.2-0.3 mm.

3. A microfluidic device for cell experiments as claimed in claim 1, characterized in that: The height of the injection groove is 0.3-0.8 mm.

4. A microfluidic device for cell experiments as claimed in claim 1, characterized in that: The pore diameter of the culture well is 0.2 mm.

5. A microfluidic device for cell experiments as claimed in claim 1, characterized in that: The surfaces of the culture tank and the injection groove are provided with a hydrophobic coating.

6. A microfluidic device for cell experiments as claimed in claim 1, characterized in that: The encapsulation layer, the liquid flow layer and the culture layer are all made of transparent materials.

7. A microfluidic device for cell experiments as claimed in claim 1, characterized in that: The number of culture tanks is no less than 6.

8. A method for cell experiment, characterized in that: The following steps are involved: S1, encapsulating the encapsulation layer, the liquid flow layer and the culture layer from top to bottom; S2. After sterilization, exhaust gas is discharged by vacuum filtration; S3, adding experimental cells into multiple groups of culture tanks in the culture layer; S4, the two ends of the automatic liquid adding device are connected to the upper sample adding hole and the upper drainage hole respectively. The liquid flow layer can realize uninterrupted gas-liquid exchange and mechanical shear force flushing through the automatic liquid adding device, providing an environment more similar to that in vivo for cell growth; S5. Flip the entire device over so that the cells can directly settle into the liquid flow layer and be quickly obtained through perfusion.

9. A cell experiment method as claimed in claim 8, characterized in that: The automatic liquid adding device comprises a peristaltic pump.

10. A cell experiment method as claimed in claim 8, characterized in that: The cell experiments include rapid in situ expansion of tumor organoid cells, rapid evaluation of multiple drugs and multiple treatment modes, acquisition of cells after evaluation, and co-culture of tumor organoids with immune cells.

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