Microfluidic device for cell experiment

Abstract

The utility model discloses a microfluidic device for a cell experiment, which comprises a packaging layer, a liquid flow layer and a culture layer, and a plurality of groups of culture grooves are arranged on the culture layer; 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 utility model provides a device which can be used for culturing tumor organs and synchronously carrying out a plurality of groups of experiments.

Description

TECHNICAL FIELD

[0001] The utility model relates to experimental device technical field, concretely is a micro -fluidic device for cell experiment. BACKGROUND

[0002] Microfluidics is the sample preparation, reaction, separation, detection and other basic operation units of biological, chemical, medical analysis process are integrated to a micron scale chip, and the whole analysis process is automatically completed. Because of its great potential in the fields of biology, chemistry, medicine, etc. It has developed into a new research field of biology, chemistry, medicine, fluid, electronics, materials, mechanics and other disciplines.

[0003] Tumor organoids are tumor cells (containing tumor stem cells) obtained by separating patient tumor tissue, which are cultured in 3D in a microenvironment similar to the in vivo by adding certain cytokines and small molecules, forming a miniature tumor model. It has similar structural characteristics and functional properties to the source tumor tissue, and can be stably amplified in a 3D culture system in vitro. Tumor organoids involve three key points: first, material selection, second, culture scheme, and third, identification scheme.

[0004] The Chinese patent with publication number CN202211584503.9 discloses a drug screening tumor organoid chip and a drug screening method. The method includes microfluidic chip preparation, double aqueous phase solution preparation, single cell suspension preparation, microfluidic manipulation, single cell loaded microgel generation, and high-throughput anti-cancer drug screening of pancreatic cancer organoids. The invention uses a microfluidic chip integrated with a normally closed pneumatic pump valve as a technical platform, a single cell suspension pre-incubated with a crosslinking agent as a chemical reaction core, and a water-in-water droplet as a forming template. Through a one-step method, single dispersed seed cells are loaded into micro-sized hydrogel carriers, achieving 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 characteristics of high-throughput liquid droplet microfluidic technology, achieving efficient and highly reproducible in vitro construction of organoids and anti-cancer drug screening, which can play a significant role in pancreatic cancer organoid-related basic research and translational applications.

[0005] The present inventors have found 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 through long-term experiments. Utility model content

[0006] The utility model overcomes these difficulties and provides a microfluidic device that can be used to culture tumor organoids and simultaneously perform multiple experiments.

[0007] To achieve the purpose, the utility model provides the following technical scheme:

[0008] The utility model provides a kind of microfluidic device for cell experiment, including from top to bottom encapsulation layer, liquid flow layer and culture layer, be equipped with multiple groups of culture tank on the culture layer, each group of culture tank includes multiple adjacent culture well;The bottom surface of the liquid flow layer is equipped with multiple groups of liquid injection groove, the liquid injection groove is arranged in the upper of the culture tank, and the liquid injection groove and the culture tank are communicated, and the both ends of the liquid injection groove are vertically equipped with multiple groups of middle layer sample addition hole and middle layer drainage hole;The encapsulation layer is equipped with multiple groups of upper layer sample addition hole and upper layer drainage hole, and the upper layer sample addition hole and the upper layer drainage hole are respectively arranged in the upper of the middle layer sample addition hole and the middle layer drainage hole.

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

[0010] In the utility model, the height of the culture well is 0.2-0.3mm, which can promote the aggregation of tumor organoids and will not settle at the bottom of the culture well. The height of the culture well is 0.2-0.3mm, which does not affect the replacement of the liquid medicine or the culture solution. The replacement of the liquid medicine or the culture solution can be realized by using uninterrupted gas-liquid exchange and mechanical shear force scouring, and the aggregation of tumor organoids in the culture well is not affected.

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

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

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

[0014] Preferably, the surface of the culture tank and the liquid injection groove is provided with a hydrophobic coating.

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

[0016] Preferably, the culture tank is not less than 6 groups.

[0017] In the utility model, the culture tank is not less than 6 groups, and each group has independent sample addition hole and drainage hole, which can be independently experimented. Especially in drug concentration screening experiment, the drug concentration can be adjusted according to the experimental progress.

[0018] The second aspect of the utility model 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, using vacuum filtration method to exhaust;

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

[0022] S4, connecting the upper layer sample hole and the upper layer drainage hole at two ends of the automatic liquid adding device respectively, and the liquid flow layer can realize uninterrupted gas-liquid exchange and mechanical shear force scouring through the automatic liquid adding device, so as to provide a more similar environment for cell growth as in vivo;

[0023] S5, turning over the whole device, and the cells can be directly settled in the liquid flow layer and quickly obtained through perfusion.

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

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

[0026] Preferably, the culture layer is provided with multiple culture tanks, each of the culture tanks comprises multiple adjacent culture holes; the bottom surface of the liquid flow layer is provided with multiple liquid injection grooves, the liquid injection grooves are arranged above the culture tanks, and the liquid injection grooves and the culture tanks are communicated, and multiple middle layer sample holes and middle layer drainage holes are vertically arranged at two ends of the liquid injection grooves; the encapsulation layer is provided with multiple upper layer sample holes and upper layer drainage holes, and the upper layer sample holes and the upper layer drainage holes are respectively arranged above the middle layer sample holes and the middle layer drainage holes.

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

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

[0029] Preferably, the aperture of the culture hole is 0.2mm.

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

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

[0032] Preferably, the culture tank is not less than 6 groups.

[0033] Compared with the prior art, the utility model has the beneficial effects and significant progress that:

[0034] 1. The microfluidic device for cell experiment of the utility model, through the unique structure design, and the height of culture hole, the height of liquid injection groove and so on parameter adjustment, make the microfluidic device for cell experiment of the utility model can simulate normal in-vivo environment, create the best experimental environment for cell, especially tumor organ culture and experiment;

[0035] 2. The microfluidic device for cell experiment of the utility model can be used for drug experiment, cell culture and acquisition experiment of different concentrations through experiment. BRIEF DESCRIPTION OF DRAWINGS

[0036] In order to more clearly illustrate the technical scheme of the utility model, the drawings required by the embodiment of the utility model will be briefly introduced below.

[0037] Obviously, the drawings in the following description are only part of the drawings in the utility model, and for the ordinary skilled person in the art, other drawings can be obtained according to these drawings without creative labor, but these other drawings also belong to the drawings required by the embodiment of the utility model.

[0038] Figure 1 It is an exploded view of the microfluidic device for cell experiment of the utility model embodiment 1;

[0039] Figure 2 It is a top view of the microfluidic device for cell experiment of the utility model embodiment 1;

[0040] Figure 3 It is a side view of the microfluidic device for cell experiment of the utility model embodiment 1;

[0041] Figure 4 It is Figure 2 It is an enlarged view of A in the middle;

[0042] Figure 5 It is a perspective view of the microfluidic device for cell experiment of the utility model embodiment 1;

[0043] Figure 6 It is a cell growth diagram in the microfluidic device of the utility model embodiment 2;

[0044] Figure 7 It is the cell drug sensitivity experiment result in the microfluidic device of the utility model embodiment 3;

[0045] Figure 8 It is the cell drug sensitivity experiment result in the microfluidic device of the utility model embodiment 3;

[0046] Figure 9The experiment result of killing tumor organoids by the chemotherapy drug in the microfluidic device co-culture system of the embodiment 5 of the utility model;

[0047] Figure 10 The experiment result of treating tumor organoids by the chemotherapy drug and the immune checkpoint inhibitor drug in the microfluidic device co-culture system of the embodiment 5 of the utility model.

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

[0049] In order to make the purpose, technical scheme, beneficial effect and significant progress of the embodiments of the utility model clearer, below, the technical scheme in the embodiments of the utility model will be clearly and completely described by combining the drawings provided in the embodiments of the utility model.

[0050] Obviously, all the described embodiments are only part of the embodiments of the utility model, not all the embodiments; all other embodiments obtained by the person skilled in the art based on the embodiments in the utility model without creative labor belong to the protection scope of the utility model.

[0051] It should be noted that the terms "first", "second" and "third" (if there are) in the specification and claims of the utility model and the drawings of the embodiments of the utility model are only used to distinguish different objects, not to describe a specific order. In addition, the term "includes" and any variations thereof are intended to cover non-exclusive inclusion. For example, the process, method, system, product or device including a series of steps or units is not limited to the listed steps or units, but optionally also includes steps or units not listed, or optionally also includes other steps or units inherent to the process, method, product or device.

[0052] It should be understood that:

[0053] In the utility model, unless otherwise explicitly specified and limited, the terms "mounting", "connection", "fixing" and the like should be understood broadly, for example, can be fixed connection, can also be detachable connection or movable connection, can also be integrated; can be direct connection, can also be indirect connection through an intermediate medium or invisible signal connection, or even optical connection, can be the internal communication of two elements or the interaction relationship of two elements, unless otherwise explicitly limited.

[0054] Those skilled in the art can understand the specific meaning of the above terms in this utility model according to the specific circumstances.

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

[0056] The technical solution of this utility model will now be described in detail with reference to specific embodiments.

[0057] Example 1

[0058] like Figures 1-5 As shown, a microfluidic device for cell experiments includes, from top to bottom, an encapsulation layer 1, a fluid flow layer 2, and a culture layer 3. The culture layer 3 has multiple sets of culture tanks 3.1, each set of culture tanks 3.1 including multiple adjacent culture wells 3.2. The bottom surface of the fluid flow layer 2 has multiple sets of injection grooves 2.3, which are located above the culture tanks 3.1 and are connected to the culture tanks 3.1. Multiple sets of middle layer sample loading holes 2.1 and middle layer drainage holes 2.2 are vertically arranged at both ends of the injection grooves 2.3. The encapsulation layer 1 has multiple sets of upper layer sample loading holes 1.1 and upper layer drainage holes 1.2, which are respectively located above the middle layer sample loading holes 2.1 and middle layer drainage holes 2.2.

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

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

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

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

[0065] The method of using the microfluidic device for cell experiments in this embodiment includes the following steps:

[0066] S1. Encapsulate the encapsulation layer 1, the fluid flow layer 2, and the culture layer 3 from top to bottom;

[0067] S2. After sterilization, exhaust the air using vacuum filtration.

[0068] Experimental cells were added to multiple culture tanks 3.1 of culture layer 3 and culture layer 3 respectively;

[0069] S4, the automatic liquid adding device is connected with the upper layer sample adding hole 1.1 and the upper layer drainage hole 1.2 respectively, and the liquid flow layer 2 can realize uninterrupted gas-liquid exchange and mechanical shear force flushing through the automatic liquid adding device, so as to provide a more similar environment to the in-vivo for cell growth;

[0070] S5, the whole device is turned over, and the cells can be directly settled in the liquid flow layer 2 and quickly obtained through perfusion.

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

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

[0073] Comparative Example 1

[0074] The device of the comparative example comprises, from top to bottom, a packaging layer, a liquid flow layer and a culture layer, the culture layer is provided with a plurality of culture grooves, each culture groove comprises a plurality of adjacent culture holes; the bottom surface of the liquid flow layer is provided with a plurality of liquid injection grooves, the liquid injection grooves are arranged above the culture grooves, and the liquid injection grooves and the culture grooves are communicated, and the two ends of the liquid injection grooves are vertically provided with a plurality of middle layer sample adding holes and middle layer drainage holes; the packaging layer is provided with a plurality 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 arranged above the middle layer sample adding holes and the middle layer drainage holes.

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

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

[0077] In this embodiment, the pore size of the culture hole is 0.5 mm.

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

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

[0080] In this embodiment, the culture grooves are not less than 6 groups.

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

[0082] The microfluidic device for cell experiment of Example 1 is used to expand and acquire human tumor organoid cells. The expansion method is briefly summarized as follows:

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

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

[0085] Step 3: Add human tumor organoid cells into the culture layer of the multi-culture tank respectively;

[0086] Step 4: Connect the upper sample adding hole and the upper drainage hole at two ends of the automatic liquid adding device respectively, and the liquid flow layer can realize uninterrupted gas-liquid exchange and mechanical shear force flushing through the automatic liquid adding device, so as to provide a more similar environment to the body for cell growth;

[0087] Step 5: Turn over the whole device, and the cells can be directly settled in the liquid flow layer, and the human tumor organoid cells can be quickly obtained through perfusion.

[0088] The cell growth in the microfluidic device is as shown in Figure 6 It can be seen that the cells grow in clusters and non-adherent growth. In summary, the microfluidic device of the utility model realizes the non-adherent culture of primary tumor cells, so that the rapid expansion of a small amount of human tumor primary cells in the microfluidic device can be realized. In addition, since the uninterrupted liquid flow system is designed on the upper part of the microfluidic device, the rapid gas-liquid exchange of the culture system can be realized, and the disadvantages of the traditional chip, such as small gap space and poor cell growth state, are overcome. In addition, after the microfluidic device body is turned over after the cells are cultured or treated, the organoid cells can be directly settled in the middle liquid flow layer, and the target cells can be quickly obtained through perfusion and used for subsequent experiments, so that the phenotype screening and molecular difference analysis of the same cell sample are realized, and the smooth development of the subsequent drug resistance mechanism is ensured.

[0089] The device of Comparative Example 1 will also be used for human tumor organoid cell expansion and acquisition, and the specific experimental method is the same as above. The results show that the cells are all gathered at the bottom of the culture layer, and the cell growth state is poor.

[0090] Example 3 Multiple drug treatment modes

[0091] The microfluidic device for cell experiment of Example 1 and the microwell plate (commercially available) are used for human tumor organoid cell expansion. The expansion method is briefly summarized as follows:

[0092] Step 1: Culture cell lines in the microwell plate and the microfluidic device respectively;

[0093] Step 2: Stimulate using the same drug and drug concentration,

[0094] Step 3: The microwell plate uses ATP or AO / PI method to detect cell activity; and the microfluidic device drug sensitivity detection platform uses fluorescence quantitative method to detect cell activity, and the differences of the two platforms are compared.

[0095] The results are shown in Figure 7 and 8 .Figure 7 These are the results of cell drug sensitivity experiments in a microfluidic device. Figure 8 The results are from a cell drug sensitivity test in a microplate. It is evident that the microfluidic device of this invention can simultaneously perform multiple experiments, and the experimental cells exhibit aggregated growth and non-adherent growth. In summary, the microfluidic device of this invention, by retaining sample loading and drainage wells and through an externally designed multi-channel system, can achieve screening of multiple drug combinations under the same culture conditions, requires a small cell volume, and allows for real-time adjustment of drug concentration based on cell response, shortening the drug sensitivity testing cycle and improving testing efficiency; it also enables medium- to long-term drug guidance, sequential drug administration protocols, and continuous efficacy evaluation.

[0096] Example 4: Drug susceptibility testing of body fluid samples

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

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

[0099] Step 2: Use an automatic liquid dispensing device to achieve automatic circulation of drug administration and provide 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) used in Example 1 for cell experiments were used to co-culture tumor organoids and immune cells, and the co-culture system was used to detect the responsiveness of targeted immunotherapy and chemoimmunotherapy.

[0103] Step 1: Culture organoids separately in microplates and microfluidic devices;

[0104] Step 2: After organoid formation, fluorescently labeled immune cells are added to the microfluidic chip at a certain target-effect cell ratio to construct a co-culture system;

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

[0106] The results are as follows Figure 9 and 10 As shown, Figure 9 The results of experiments on the killing of tumor organoids by chemotherapeutic drugs in a microfluidic co-culture system are presented. Figure 10 This presents experimental results of the combined treatment of tumor organoids with chemotherapeutic drugs and immune checkpoint inhibitors in a microfluidic co-culture system.Figure 9 It is shown that immune cells can enhance the killing effect of low-dose chemotherapy drugs on tumor organoids. Figure 10 It is shown that the microfluidic device of the utility model can be used for evaluating the killing effect of immune checkpoint inhibitors combined with other drugs on tumor organoids.

[0107] In summary, the microfluidic device of the utility model can construct a tumor organoid immune microenvironment by adding immune cells, so that it can be applied to multidimensional drug combination therapy efficacy evaluation, and the application scenarios of the microfluidic chip for guiding clinical drug selection are enriched.

[0108] In the description process of the above description:

[0109] The description of the terms "the embodiment", "the utility model embodiment", "as shown", "further", "further improved technical scheme" and the like means that the specific features, structures, materials or characteristics described in the embodiment or example are contained in at least one embodiment or example of the utility model; in the description, the illustrative description of the above terms is not necessarily for the same embodiment or example, and the specific features, structures, materials or characteristics described can be combined or combined in any one or more embodiments or examples in a suitable manner; in addition, without contradiction, ordinary skilled in the art can combine or combine the different embodiments or examples described in the description and the characteristics of different embodiments or examples.

[0110] Finally, it should be pointed out that:

[0111] The above embodiments are only used to illustrate the technical solutions of the utility model, but not limit them;

[0112] Although the utility model has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that it can still modify the technical solutions recorded in the foregoing embodiments, or make equivalent replacement to part or all of the technical features, and these modifications or replacements do not make the essence of the corresponding technical solution deviate from the scope of the technical solutions of the embodiments of the utility model, and the non-essential improvements and adjustments or replacements made by those skilled in the art according to the content of the description all belong to the scope of the utility model claimed.

Claims

1. A microfluidic device for cell experiments, characterized in that, Including, from top to bottom, the encapsulation layer, the fluid flow layer, and the culture layer. The culture layer is provided with multiple sets of culture tanks, and each set of culture tanks includes multiple adjacent culture wells; The bottom surface of the liquid flow layer is provided with multiple sets of liquid injection grooves. The liquid injection grooves are located above the culture tank and are connected to the culture tank. Multiple sets of middle layer sample addition holes and middle layer drainage holes are vertically provided at both ends of the liquid injection grooves. The encapsulation layer is provided with multiple sets of upper layer sample feeding holes and upper layer drainage holes, which are respectively arranged above the middle layer sample feeding holes and the middle layer drainage holes.

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

3. The microfluidic device for cell experiments as described 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 described in claim 1, characterized in that, The pore size of the culture well is 0.2 mm.

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

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

7. A microfluidic device for cell experiments as described in claim 1, characterized in that, There shall be no fewer than 6 sets of culture tanks.

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