Microfluidic chip for organoid culture and detection and application thereof

Abstract

This invention provides a microfluidic chip for organoid culture and detection, and its application. The microfluidic chip includes a microvalve control layer, a microvalve film layer, and a processing layer. The microvalve film layer is disposed between the microvalve control layer and the processing layer, and adjacent layers are sealed together. The microvalve control structure includes independent gas channels that cooperate with the microvalve film layer. On the side of the processing layer facing the microvalve film layer, there are organoid culture structures, single-cell capture and amplification structures, and microvalve control regions. The organoid culture structure and the single-cell capture and amplification structure each have multiple microvalve control regions. The organoid culture structure and the single-cell capture and amplification structure are connected. One technical advantage of this invention is its rational design, enabling the culture and detection of organoids and facilitating accurate analysis and detection of single cells.

Description

Technical Field

[0001] This invention belongs to the field of microchannel fluid control technology, specifically relating to a microfluidic chip for organoid culture and detection and its applications. Background Technology

[0002] Organoids are cell clusters generated by culturing human organ stem cells in vitro, producing cells that resemble organs in vivo. As a 3D in vitro cell culture technology, organoids can stably deliver the genome and characteristics of tumors while preserving the heterogeneity of individual samples. Studies have shown that organoids can encompass 97% of gene mutations in tumors. Organoid technology has, for the first time, offered the possibility of validating the actual efficacy of cancer drugs in vitro, thus becoming one of the most watched cutting-edge technologies in the field of precision cancer medicine. However, during organoid culture, especially during transfer, organoid cell clusters are susceptible to mechanical damage. Furthermore, organoid clusters are prone to fusion during culture, making localized observation difficult. Insufficient sample size also prevents high-throughput drug sensitivity testing of organoids on well plates.

[0003] Furthermore, current analyses of organoids largely involve simply observing the size of organoid clusters, determining the viability of cell clusters, or studying organoid populations. Since cells are the basic units of life, studies based on cell populations can, to some extent, obscure important information about individual cells or a small number of cells.

[0004] Therefore, in the current process of organoid culture and analysis, it is difficult to achieve localized observation because organoid clusters are prone to fusion, and it is also not conducive to accurate analysis and research of individual cells. Summary of the Invention

[0005] The present invention aims to at least solve one of the technical problems existing in the prior art, and to provide a new technical solution for a microfluidic chip for organoid culture and detection and its application.

[0006] According to a first aspect of this application, a microfluidic chip for organoid culture and detection is provided, comprising a microvalve control layer, a microvalve film layer and a processing layer, wherein the microvalve film layer is disposed between the microvalve control layer and the processing layer, and adjacent layers are sealed together. The microvalve control layer is provided with a first liquid inlet, a first liquid outlet, a digestive fluid inlet, a second liquid inlet, a cell suspension outlet, an amplification product outlet, a microvalve air inlet, and a microvalve control structure; wherein the first liquid inlet, the first liquid outlet, the digestive fluid inlet, the second liquid inlet, the cell suspension outlet, and the amplification product outlet all penetrate into the processing layer; the microvalve control structure includes independent gas channels, each gas channel being connected to a microvalve air inlet for controlling the gas pressure within the gas channel; the gas channels cooperate with the microvalve film layer to control the opening or closing of each microvalve control area; The processing layer has an organoid culture structure, a single-cell capture and amplification structure, and a microvalve control region on the side facing the microvalve film layer. The organoid culture structure and the single-cell capture and amplification structure each have multiple microvalve control regions. The organoid culture structure is connected to the single-cell capture and amplification structure. The organoid culture structure is connected to the first liquid inlet, the first liquid outlet, and the digestive fluid inlet. The single-cell capture and amplification structure is connected to the second liquid inlet, the cell suspension outlet, and the amplification product outlet. By controlling the opening or closing of the corresponding micro-valve control area, the gel mixture formed by mixing tumor cells and gel can enter the organoid culture structure through the first liquid inlet to culture the tumor cells, and can be discharged through the first liquid outlet; organoid digestive enzymes can enter the organoid culture structure through the digestive fluid inlet and digest the organoid mass into single cells to form a single-cell suspension; the single-cell suspension enters the single-cell capture and amplification structure from the organoid culture structure to capture single cells; the cell lysis solution entering through the second liquid inlet lyses the single cells to form cell lysis fluid, and then the amplification enzyme solution entering the reaction fluid channel through the second liquid inlet amplifies the cell lysis fluid to form amplification products, which can be discharged through the amplification product outlet.

[0007] Optionally, the gas channel includes a first gas channel, a second gas channel, a third gas channel, a fourth gas channel, a fifth gas channel, a sixth gas channel, and a seventh gas channel; The first gas channel is equipped with a control microvalve A; the second gas channel is equipped with a control microvalve B; the third gas channel is equipped with control microvalve C, D, and E; the fourth gas channel is equipped with control microvalve F and G; the fifth gas channel is equipped with a control microvalve H; the sixth gas channel is equipped with a control microvalve I; and the seventh gas channel is equipped with a control microvalve J. Each control microvalve corresponds to a microvalve control area on the processing layer, and each control microvalve cooperates with the microvalve film layer to control the opening or closing of each microvalve control area. The organoid culture structure includes a first liquid channel, an organoid culture unit, and a digestive fluid channel, wherein one end of the first liquid channel is connected to a first liquid inlet, and the other end is connected to a first liquid outlet; the digestive fluid channel is connected to the digestive fluid inlet. The organoid culture unit includes an organoid culture chamber, a digestion mixing zone, and an organoid lysis chamber. The organoid culture chamber is connected to a first liquid channel and a digestive fluid channel, respectively. The first liquid channel is provided with a microvalve control area A for controlling the opening or closing of the first liquid channel. The digestive fluid channel is provided with a microvalve control area B for controlling the opening or closing of the digestive fluid channel. When the A microvalve control area is opened, the gel mixture formed by the mixing of tumor cells and gel enters the first liquid channel through the first liquid inlet and then enters the organoid culture chamber. Culture medium is then injected into the first liquid channel through the first liquid inlet to culture the tumor cells. When microvalve A is closed and microvalve B is opened, the organoid digestive enzymes that enter the digestive fluid channel through the digestive fluid inlet pass through the organoid culture chamber and the digestion mixing zone in sequence and enter the organoid lysis chamber. The gel mixture mixes with the organoid digestive enzymes in the digestion mixing zone and digests the organoid mass into single cells in the organoid lysis chamber to form a single-cell suspension in the organoid lysis chamber. The single-cell capture and amplification structure includes a main channel, a reaction liquid channel, and a cell processing unit. Each cell processing unit includes a cell lysis chamber, a lysis termination chamber, and an amplification chamber. One end of the main channel is connected to the organoid lysis chamber, and the other end is connected to the cell suspension outlet. The main channel includes a capture region and a bypass region, which are connected in parallel. The capture region includes an inlet end, a capture segment, a connecting segment, and an outlet end, which are sequentially connected. The bypass region, the cell lysis chamber, the termination lysis chamber, the amplification chamber, and the amplification product outlet are sequentially connected, and an F microvalve control area is provided between the bypass region and the cell lysis chamber. An H microvalve control area is provided between the cell lysis chamber and the termination lysis chamber. An I microvalve control area is provided between the termination lysis chamber and the amplification chamber. A G microvalve control area is provided between the amplification chamber and the amplification product outlet. The main channel is provided with a C-microvalve control area, a D-microvalve control area, and an E-microvalve control area. The C-microvalve control area is located at the inlet end of the main channel near the bypass area and the capture area, and is used to control the opening or closing of the main channel. The D-microvalve control area is located in the bypass area, and is situated between the outlet end of the bypass area and the connection point between the bypass area and the cell lysis chamber, and is used to control the opening or closing of the bypass area. The E-microvalve control area is located at the outlet end of the main channel near the bypass area and the capture area, and is used to control the opening or closing of the main channel. One end of the reaction liquid flow channel is connected to the second liquid inlet, and the other end is connected to the capture area. The connection point between the reaction liquid flow channel and the capture area is located in the connecting section. A sixth control valve area is provided on the reaction liquid flow channel to control the opening or closing of the reaction liquid flow channel. Opening the sixth control valve area closes the C micro valve control area, the D micro valve control area, and the E micro valve control area. When the F microvalve control area and the G microvalve control area are closed, the single-cell suspension enters the main channel from the digestive fluid channel. When it flows through the capture area, the single cell is captured and the capture area is blocked. Then the single-cell suspension flows from the bypass area to the cell suspension outlet. When the C microvalve control area and the D microvalve control area are closed, and the F microvalve control area and the G microvalve control area are opened, the cell lysis solution entering through the second liquid inlet passes through the reaction liquid channel and the capture area, carrying the captured single cells through the bypass area and into the cell lysis chamber. The F and H microvalve control areas are closed, and single cells lyse in the cell lysis chamber. The F and H microvalve control areas are then opened, allowing the termination lysis solution entering the reaction liquid channel through the second liquid inlet to flow into the termination lysis chamber along with the liquid in the cell lysis chamber. The F and I microvalve control areas are then closed, allowing the termination lysis solution to mix thoroughly with the cell lysis liquid, thus terminating lysis. The F and I microvalve control areas are then opened, allowing the amplification enzyme solution entering the reaction liquid channel through the second liquid inlet to flow into the amplification chamber along with the termination lysis solution in the termination lysis chamber. The F and J microvalve control areas are then closed, and amplification begins. After a preset time, amplification is terminated to form amplification products. Finally, the F and J microvalve control areas are opened, allowing purified water entering the reaction liquid channel through the second liquid inlet to discharge the amplification products from the amplification product outlet.

[0008] Optionally, multiple cell processing units are arranged in parallel, each cell processing unit corresponding to a capture area and a bypass area, and an E-microvalve control area is provided between adjacent capture areas.

[0009] Optionally, there are multiple organoid culture units and multiple digestive fluid channels. The multiple organoid culture units are connected in series through the first liquid channel, and the A microvalve control area is set between adjacent organoid culture units. Each organoid culture unit corresponds to one digestive fluid channel.

[0010] Optionally, the microvalve control layer, the microvalve film layer, and the processing layer are all made of PDMS material, and the microvalve control layer and the microvalve film layer are bonded by oxygen plasma assisted bonding, and the microvalve film layer and the processing layer are bonded by oxygen plasma assisted bonding.

[0011] Optionally, the microvalve film layer is spin-coated from PDMS material and has a thickness of 15 micrometers.

[0012] Optionally, the main channel is bent multiple times to form multiple interconnected sub-channels, each of which is connected to multiple cell processing units.

[0013] Optionally, the reaction liquid channel includes multiple sub-reaction liquid channels, one end of each sub-reaction liquid channel is connected to the second liquid inlet, and the other end is connected to a capture area.

[0014] According to a second aspect of this application, an application of a microfluidic chip for organoid culture and detection is provided, applied to the microfluidic chip as described in the first aspect, comprising: A lesion sample is obtained from the patient, and tumor cells are obtained by processing the sample with digestive fluid. The tumor cells are then mixed with a gel to obtain a gel mixture. The gel mixture was injected into the organoid culture chamber, and the chamber was placed in an incubator to fix the gel; then, culture medium was introduced into the organoid culture chamber for culturing. Organoid digestive enzymes are introduced into the organoid culture chamber to digest the organoid mass into single cells and form a single-cell suspension; then, a digestion-terminating solution is introduced into the single-cell suspension to stop digestion. Capture single cells in a single-cell suspension; Single cells are lysed using a cell lysis buffer to form a cell lysis liquid; then, the cell lysis liquid is amplified using an amplification enzyme solution to form an amplification product.

[0015] Optionally, the ratio of tumor cells to gel is 1:2; The organoid digestive enzyme used was trypsin, and the digestion termination solution was PBS buffer.

[0016] One technical advantage of this invention is that: In the embodiments of this application, by realizing organoid culture, single-cell capture, lysis and amplification on a microfluidic chip, it is possible not only to effectively avoid mechanical damage to organoid cell clusters during organoid transfer and better protect organoid cell clusters, but also to accurately analyze and detect single cells to obtain the heterogeneity between tumor organoid cells.

[0017] In addition, this microfluidic chip is based on microfluidic technology and micro-nano fabrication technology, which can achieve structural miniaturization, sample miniaturization and fluid precision. It can precisely control liquids in micron-level channels, enabling not only high-throughput culture and precise control of organoids, but also accurate single-cell capture, lysis and gene amplification of organoid cells. Attached Figure Description

[0018] Figure 1 This is an exploded structural diagram of a microfluidic chip for organoid culture and detection according to an embodiment of the present invention. Figure 2 This is a schematic diagram of the microvalve control layer of a microfluidic chip for organoid culture and detection according to an embodiment of the present invention. Figure 3 for Figure 2 Enlarged detail image of section M in the middle; Figure 4 This is a schematic diagram of the processing layer of a microfluidic chip for organoid culture and detection according to an embodiment of the present invention. Figure 5 for Figure 4 Enlarged detail image of point N in the middle; Figure 6 This is a schematic diagram of the connection relationship at the single-cell capture site of a microfluidic chip used for organoid culture and detection according to an embodiment of the present invention. Figure 7 This is an enlarged schematic diagram of the single-cell capture area of ​​a microfluidic chip used for organoid culture and detection according to an embodiment of the present invention. Figure 8 This is a schematic diagram of the organoid culture process of a microfluidic chip for organoid culture and detection according to an embodiment of the present invention. Figure 9 This is a schematic diagram of the organoid digestion process of a microfluidic chip for organoid culture and detection according to an embodiment of the present invention. Figure 10 This is a schematic diagram of the single-cell injection and capture region of a microfluidic chip for organoid culture and detection according to an embodiment of the present invention. Figure 11This is a schematic diagram of the structure of a microfluidic chip for organoid culture and detection according to an embodiment of the present invention, showing a single cell flowing from the capture region into the cell lysis chamber. Figure 12 This is a schematic diagram of the single-cell lysis process of a microfluidic chip for organoid culture and detection according to an embodiment of the present invention. Figure 13 This is a schematic diagram of a single cell entering the termination lysis chamber of a microfluidic chip for organoid culture and detection according to an embodiment of the present invention. Figure 14 This is a schematic diagram of a single cell entering the amplification chamber of a microfluidic chip for organoid culture and detection, according to an embodiment of the present invention.

[0019] In the diagram: 1. Microvalve control layer; 101. First liquid inlet; 102. First liquid outlet; 103. Digestion fluid inlet; 104. Second liquid inlet; 105. Cell suspension outlet; 106. Amplification product outlet; 107. First gas channel; 1071. First microvalve inlet; 108. Second gas channel; 1081. Second microvalve inlet; 109. Third gas channel; 1091. Third microvalve inlet; 110. Fourth gas channel; 1101. Fourth microvalve inlet; 111. Fifth gas channel; 1111. Fifth microvalve inlet; 112. Sixth gas channel; 1121. Sixth microvalve inlet; 113. Seventh gas channel; 1131. Seventh microvalve inlet; 2. Microvalve thin film layer; 3. Processing layer; 301. First liquid channel; 302. Digestive fluid channel; 303. Organoid culture chamber; 304. Digestion mixing zone; 305. Organoid lysis chamber; 306. Main channel; 3061. Capture region; 30611. Capture segment; 30612. Connecting segment; 3062. Bypass region; 307. Reaction fluid channel; 308. Cell lysis chamber; 309. Termination lysis chamber; 310. Amplification chamber. Detailed Implementation

[0020] Various exemplary embodiments of the present application will now be described in detail with reference to the accompanying drawings. It should be noted that, unless otherwise specifically stated, the relative arrangement, numerical expressions, and values ​​of the components and steps set forth in these embodiments do not limit the scope of the present application.

[0021] The embodiments of this application will now be described in detail. Examples of these embodiments are illustrated in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain this application, and should not be construed as limiting this application. All other embodiments obtained by those skilled in the art based on the embodiments of this application without inventive effort are within the scope of protection of this application.

[0022] See Figures 1 to 14 As shown, according to a first aspect of this application, a microfluidic chip for organoid culture and detection is provided.

[0023] join Figure 1 The microfluidic chip includes a microvalve control layer 1, a microvalve film layer 2, and a processing layer 3. The microvalve film layer 2 is disposed between the microvalve control layer 1 and the processing layer 3, and adjacent layers are sealed together. For ease of observation, the microvalve control layer 1, the microvalve film layer 2, and the processing layer 3 are all made of transparent material.

[0024] In one specific embodiment, the thickness of the microvalve thin film layer 2 is 12 micrometers to 30 micrometers, preferably 20 micrometers, which not only helps to achieve the seal between the microvalve control layer 1 and the processing layer 3, but also helps to reduce the weight of the microfluidic chip.

[0025] Specifically, see Figure 2 and Figure 3 The microvalve control layer 1 is provided with a first liquid inlet 101, a first liquid outlet 102, a digestive fluid inlet 103, a second liquid inlet 104, a cell suspension outlet 105, an amplification product outlet 106, a microvalve air inlet, and a microvalve control structure. The first liquid inlet 101, the first liquid outlet 102, the digestive fluid inlet 103, the second liquid inlet 104, the cell suspension outlet 105, and the amplification product outlet 106 all extend into the processing layer 3. The microvalve control structure includes independent gas channels, each gas channel connected to a microvalve air inlet for controlling the gas pressure within the gas channel. The gas channels cooperate with the microvalve film layer 2 to control the opening or closing of each microvalve control area.

[0026] It should be noted that each microvalve air inlet of the microvalve control layer 1 is connected to an air pump to achieve digital control, thereby enabling the switching of the on / off state of the microvalve control structure of the microfluidic chip to control the response of the microfluidic chip.

[0027] For more details, see Figure 4 and Figure 5The processing layer 3 is provided with an organoid culture structure, a single-cell capture and amplification structure, and a microvalve control region on the side facing the microvalve film layer 2. The organoid culture structure and the single-cell capture and amplification structure are each provided with a plurality of microvalve control regions. The organoid culture structure is connected to the single-cell capture and amplification structure. The organoid culture structure is connected to the first liquid inlet 101, the first liquid outlet 102, and the digestive fluid inlet 103, respectively. The single-cell capture and amplification structure is connected to the second liquid inlet 104, the cell suspension outlet 105, and the amplification product outlet 106, respectively.

[0028] In this application, by controlling the opening or closing of the corresponding microvalve control area, the gel mixture formed by mixing tumor cells and gel can enter the organoid culture structure through the first liquid inlet 101 to culture the tumor cells, and can be discharged through the first liquid outlet 102; the organoid digestive enzyme can enter the organoid culture structure through the digestive fluid inlet 103 and digest the organoid mass into single cells to form a single-cell suspension; the single-cell suspension enters the single-cell capture and amplification structure from the organoid culture structure to capture single cells; the cell lysis solution entering through the second liquid inlet 104 lyses the single cells to form cell lysis fluid, and then the amplification enzyme solution entering through the second liquid inlet 104 amplifies the cell lysis fluid to form amplification products, and can be discharged through the amplification product outlet 106.

[0029] It should be noted that, for ease of understanding, Figure 2 In this context, A refers to valve A controlling the microvacuum, and B refers to valve B controlling the microvacuum. Figure 3 In this context, C refers to C-controlled microvalves, D refers to D-controlled microvalves, E refers to E-controlled microvalves, F refers to F-controlled microvalves, G refers to G-controlled microvalves, H refers to H-controlled microvalves, I refers to I-controlled microvalves, and J refers to J-controlled microvalves.

[0030] Figure 4 In this context, A' refers to microvalve control area A, and B' refers to microvalve control area B. Figure 5 In this context, C' refers to the C microvalve control area, D' refers to the D microvalve control area, E' refers to the E microvalve control area, F' refers to the F microvalve control area, G' refers to the G microvalve control area, H' refers to the H microvalve control area, I' refers to the I microvalve control area, and J' refers to the J microvalve control area.

[0031] Furthermore, in Figures 8 to 14 To facilitate understanding of the cooperation between the microvalve control layer 1 and the processing layer 3, the various processes of a single cell are illustrated by showing how the control microvalve directly covers the microvalve control layer 1. In the diagram, the control microvalve is black, indicating that the microvalve control area covered by the control microvalve is in the closed state. The rest represent the microvalve control area covered by the control microvalve in the open state.

[0032] Optionally, such as Figure 2 As shown, the gas channels include a first gas channel 107, a second gas channel 108, a third gas channel 109, a fourth gas channel 110, a fifth gas channel 111, a sixth gas channel 112, and a seventh gas channel 113; the micro-valve inlets include a first micro-valve inlet 1071, a second micro-valve inlet 1081, a third micro-valve inlet 1091, a fourth micro-valve inlet 1101, a fifth micro-valve inlet 1111, a sixth micro-valve inlet 1121, and a seventh micro-valve inlet 1131. Specifically, the first micro-valve inlet 1071 is connected to the first gas channel 107; the second micro-valve inlet 1081 is connected to the second gas channel 108; the third micro-valve inlet 1091 is connected to the third gas channel 109; the fourth micro-valve inlet 1101 is connected to the fourth gas channel 110; the fifth micro-valve inlet 1111 is connected to the fifth gas channel 111; the sixth micro-valve inlet 1121 is connected to the sixth gas channel 112; and the seventh micro-valve inlet 1131 is connected to the seventh gas channel 113.

[0033] like Figure 3 As shown, the first gas channel 107 is equipped with a control microvalve A; the second gas channel 108 is equipped with a control microvalve B; the third gas channel 109 is equipped with control microvalves C, D, and E; the fourth gas channel 110 is equipped with control microvalves F and G; the fifth gas channel 111 is equipped with a control microvalve H; the sixth gas channel 112 is equipped with a control microvalve I; and the seventh gas channel 113 is equipped with a control microvalve J. Each control microvalve corresponds to a microvalve control area on the processing layer 3, and each control microvalve cooperates with the microvalve film layer 2 to control the opening or closing of each microvalve control area. like Figure 4 As shown, see also Figure 8 and Figure 9 The organoid culture structure includes a first liquid channel 301, an organoid culture unit, and a digestive fluid channel 302. One end of the first liquid channel 301 is connected to a first liquid inlet 101, and the other end is connected to a first liquid outlet 102. The digestive fluid channel 302 is connected to the digestive fluid inlet 103. See Figure 4 , Figure 5 , Figures 10 to 14The organoid culture unit includes an organoid culture chamber 303, a digestion mixing zone 304, and an organoid lysis chamber 305. The organoid culture chamber 303 is connected to a first liquid channel 301 and a digestive fluid channel 302. The first liquid channel 301 is provided with a microvalve control area A, which is used to control the opening or closing of the first liquid channel 301 to prevent the inflow of culture medium from affecting the digestion process. The digestive fluid channel 302 is provided with a microvalve control area B, which is used to control the opening or closing of the digestive fluid channel 302 to select the digestive fluid channel 302 to flow through during the digestion process. like Figure 1 As shown, when the A microvalve control area is opened, the gel mixture formed by the mixing of tumor cells and gel enters the first liquid channel 301 through the first liquid inlet 101 and then enters the organoid culture chamber 303. Culture medium is then injected into the first liquid channel 301 through the first liquid inlet 101 to culture the tumor cells. like Figure 2 As shown, the A microvalve control area is closed and the B microvalve control area is opened. The organoid digestive enzymes entering the digestive fluid flow channel 302 through the digestive fluid inlet 103 pass through the organoid culture chamber 303 and the digestion mixing zone 304 in sequence and enter the organoid lysis chamber 305. The gel mixture and the organoid digestive enzymes are mixed in the digestion mixing zone 304 and digest the organoid mass into single cells in the organoid lysis chamber 305 to form a single-cell suspension in the organoid lysis chamber 305. See Figure 4 as well as Figures 10 to 14 The single-cell capture and amplification structure includes a main channel 306, a reaction liquid channel 307, and a cell processing unit. Each cell processing unit includes a cell lysis chamber 308, a lysis termination chamber 309, and an amplification chamber 310. One end of the main channel 306 is connected to the organoid lysis chamber 305, and the other end is connected to the cell suspension outlet 105. Figure 6As shown, the main channel 306 includes a capture region 3061 and a bypass region 3062. The capture region 3061 and the bypass region 3062 are connected in parallel. The capture region 3061 includes an inlet end, a capture segment 30611, a connecting segment 30612, and an outlet end. The inlet end, capture segment 30611, connecting segment 30612, and outlet end of the capture region 3061 are connected sequentially. The diameter of the capture segment 30611 is smaller than the diameter of a single cell, thereby achieving the capture of single cells. The bypass region 3062, the... The cell lysis chamber 308, the termination lysis chamber 309, the amplification chamber 310, and the amplification product outlet 106 are sequentially connected, and an F microvalve control area is provided between the bypass region 3062 and the cell lysis chamber 308; an H microvalve control area is provided between the cell lysis chamber 308 and the termination lysis chamber 309; an I microvalve control area is provided between the termination lysis chamber 309 and the amplification chamber 310; and a G microvalve control area is provided between the amplification chamber 310 and the amplification product outlet 106. The main channel 306 is provided with a C microvalve control area, a D microvalve control area, and an E microvalve control area. The C microvalve control area is located at the inlet end of the main channel 306 near the bypass region 3062 and the capture region 3061, and is used to control the opening or closing of the main channel 306. The D microvalve control area is located in the bypass region 3062, and is located between the outlet end of the bypass region 3062 and the connection point between the bypass region 3062 and the cell lysis chamber 308, and is used to control the opening or closing of the bypass region 3062. The E microvalve control area is located at the outlet end of the main channel 306 near the bypass region 3062 and the capture region 3061, and is used to control the opening or closing of the main channel 306. One end of the reaction liquid flow channel 307 is connected to the second liquid inlet 104, and the other end is connected to the capture area 3061. The connection point between the reaction liquid flow channel 307 and the capture area 3061 is located in the connecting section 30612. A sixth control valve area is provided on the reaction liquid flow channel 307 for controlling the opening or closing of the reaction liquid flow channel 307. Opening the sixth control valve area closes the C micro-valve control area, the D micro-valve control area, and the E micro-valve control area. See Figure 6 , Figure 7 , Figure 10 When the F microvalve control area and the G microvalve control area are closed, the single-cell suspension enters the main channel 306 from the digestive fluid channel 302. When it flows through the capture area 3061, the single cell is captured and the capture area 3061 is blocked. Then the single-cell suspension flows from the bypass area 3062 to the cell suspension outlet 105. See Figure 11Close the C microvalve control area and the D microvalve control area, open the F microvalve control area and the G microvalve control area, and the cell lysis solution entering through the second liquid inlet 104 passes through the reaction liquid channel 307, the capture area 3061, and carries the captured single cells through the bypass area 3062 into the cell lysis chamber 308. See Figure 12 The F microvalve control area and the H microvalve control area are closed, and single cells lyse in the cell lysis chamber 308; [the cells then participate in...] Figure 13 Opening the F microvalve control area and the H microvalve control area allows the termination lysis solution entering the reaction liquid flow channel 307 through the second liquid inlet 104 to carry the liquid in the cell lysis chamber 308 into the termination lysis chamber 309. Then, closing the F microvalve control area and the I microvalve control area allows the termination lysis solution to mix thoroughly with the cell lysis liquid, thus terminating the lysis process. (See also...) Figure 14 Open the F microvalve control area and the I microvalve control area. The amplification enzyme solution entering the reaction liquid flow channel 307 through the second liquid inlet 104 carries the termination lysis solution in the termination lysis chamber 309 into the amplification chamber 310. After the amplification chamber 310 is full, close the F microvalve control area and the J microvalve control area to perform amplification. After a preset time, the amplification is terminated to form an amplification product. For example, the chip is placed at 30°C for 2 hours, and then placed at 65°C for 5 minutes to terminate the amplification. Open the F microvalve control area and the J microvalve control area. The purified water entering the reaction liquid flow channel 307 through the second liquid inlet 104 discharges the amplification product from the amplification product outlet 106.

[0034] In the above embodiments, the microfluidic chip has a reasonable structural design, which can simultaneously realize various processes such as organoid culture, organoid lysis, single-cell capture, single-cell lysis, and single-cell amplification, thereby enabling accurate analysis and detection of single cells to obtain the heterogeneity between tumor organoid cells.

[0035] In one specific embodiment, the specific dimensions can be designed according to the required cell size. Preferably, the diameter of the main channel 306 and the channel through which a single cell can pass is 30 micrometers to 40 micrometers, and the diameter of the trapping section 30611 is 4 micrometers to 8 micrometers. More preferably, the diameter of the main channel 306 and the channel through which a single cell can pass is 30 micrometers, and the diameter of the trapping section 30611 is 5 micrometers.

[0036] For example, by providing a groove structure on the side of the microvalve control layer 1 near the microvalve film layer 2, the groove structure cooperates with the microvalve film layer 2 to form various gas channels, thereby enabling the opening or closing of each microvalve control area on the processing layer 3. The width of the groove structure is 20 micrometers to 100 micrometers, and the height is 10 micrometers to 100 micrometers; the thickness of the microvalve film layer 2 is 10 micrometers to 50 micrometers.

[0037] Similarly, a groove structure is also provided on the side of the processing layer 3 near the microvalve film layer 2, and the groove structure and the microvalve film layer 2 form various chambers and channels. The height of the first liquid channel 301 of the processing layer 3 is 10 micrometers to 200 micrometers, and the width is 50 micrometers to 2000 micrometers; the height of the digestive fluid channel 302 is 15 micrometers to 100 micrometers, and the width is 50 micrometers to 2000 micrometers; the width of the reaction fluid channel 307 is consistent with the channel that can pass through a single cell. Cell lysis chamber 308 is a cuboid structure with a height of 15-100 micrometers, a length of 200-2000 micrometers, and a width of 100-1000 micrometers; termination lysis chamber 309 is a cuboid structure with a height of 15-100 micrometers, a length of 200-2000 micrometers, and a width of 100-1000 micrometers; amplification chamber 310 is a cuboid structure with a height of 15-100 micrometers, a length of 400-4000 micrometers, and a width of 200-2000 micrometers.

[0038] Optionally, multiple cell processing units are arranged in parallel, each cell processing unit corresponding to a capture area 3061 and a bypass area 3062, and an E-microvalve control area is provided between adjacent capture areas 3061.

[0039] In the above embodiments, multiple single cells can be captured simultaneously, which is beneficial to improve the capture efficiency of single cells, thereby better realizing the single cell lysis and single cell amplification process, enabling accurate analysis and detection of single cells, and helping to accurately obtain the heterogeneity between tumor organoid cells.

[0040] Optionally, there are multiple organoid culture units and multiple digestive fluid channels 302. Multiple organoid culture units are connected in series through the first liquid channel 301, and the A microvalve control area is set between adjacent organoid culture units. Each organoid culture unit corresponds to one digestive fluid channel 302.

[0041] In the above embodiments, multiple organoid culture units can culture organoids separately, which significantly improves the culture efficiency of organoids. Simultaneously, the digestive fluid channel 302 allows for the digestion and lysis of organoids in each culture unit, effectively breaking them down into single cells. This facilitates the subsequent single-cell capture process. The structure is simple and the operation is very convenient.

[0042] Optionally, the microvalve control layer 1, the microvalve film layer 2, and the processing layer 3 are all made of PDMS (polydimethylsiloxane) material, and the microvalve control layer 1 and the microvalve film layer 2 are bonded by oxygen plasma assisted bonding, and the microvalve film layer 2 and the processing layer 3 are bonded by oxygen plasma assisted bonding.

[0043] In the above embodiments, the materials of the microvalve control layer 1, the microvalve film layer 2, and the processing layer 3 help to realize the functions of the microfluidic chip. Moreover, the use of oxygen plasma-assisted bonding to achieve sealing between the microvalve control layer 1 and the microvalve film layer 2, as well as between the microvalve film layer 2 and the processing layer 3, results in a good sealing effect and simple processing.

[0044] Optionally, the microvalve film layer 2 is spin-coated from PDMS material with a thickness of 15 micrometers. This facilitates rapid processing of the microvalve film layer 2 and also helps the microvalve film layer 2 cooperate with the microvalve control layer 1 to form various gas flow channels, thereby enabling the opening or closing of each microvalve control area of ​​the processing layer 3, which is beneficial to the realization of various functions of the processing layer 3.

[0045] Optionally, the main channel 306 is bent multiple times to form multiple interconnected sub-channels, each sub-channel being connected to multiple cell processing units.

[0046] In the above embodiments, the main channel 306 has a reasonable structural design, which is conducive to connecting multiple cell processing units at the same time, thereby improving the efficiency of single cell capture and processing.

[0047] Optionally, the reaction liquid channel 307 includes a plurality of sub-reaction liquid channels 307, one end of each sub-reaction liquid channel 307 is connected to the second liquid inlet 104, and the other end is connected to a capture region 3061.

[0048] In the above embodiments, the reaction liquid flow channel 307 is reasonably designed to simultaneously introduce cell lysis solution into multiple cell lysis chambers 308, thereby realizing the lysis process of multiple single cells. It can also simultaneously introduce termination lysis solution into multiple termination lysis chambers 309, thereby realizing the termination lysis process of multiple single cells. Furthermore, it can introduce amplification enzyme solution into multiple amplification chambers 310, thereby realizing the amplification process of multiple single cells.

[0049] According to a second aspect of this application, an application of a microfluidic chip for organoid culture and detection is provided, applied to the microfluidic chip as described in the first aspect, comprising: A lesion sample is obtained from the patient, and tumor cells are obtained by processing the sample with digestive fluid. The tumor cells are then mixed with a gel to obtain a gel mixture; for example, the tumor cells can be gastric cancer cells.

[0050] A gel mixture is injected into the organoid culture chamber 303, and the gel is fixed in an incubator, for example, by placing the chip in a 37°C incubator for half an hour to fix the gel. Then, sterile gas is injected through the first liquid inlet 101 to expel any remaining gel mixture in the first liquid channel 301. Next, culture medium is introduced into the organoid culture chamber 303 for culturing. For example, the gel mixture can be injected through the first liquid inlet 101, and the gel mixture can be injected into the organoid culture chamber 303 under the influence of gravity.

[0051] Organoid digestive enzymes are introduced into organoid culture chamber 303 to digest the organoid mass into single cells and form a single-cell suspension; then, a digestion-terminating solution is introduced into the single-cell suspension to terminate digestion.

[0052] Capture single cells in a single-cell suspension.

[0053] Single cells are lysed using cell lysis buffer to form cell lysis fluid. Cell lysis buffer is continuously injected until the cell lysis chamber 308 is filled. For example, the chip is placed in an environment of 65 degrees Celsius for 10 minutes to lyse the cells. The cell lysis fluid is then amplified by amplification enzyme solution to form amplification products.

[0054] Optionally, the ratio of tumor cells to gel is 1:2; The organoid digestive enzyme used was trypsin, and the digestion termination solution was PBS buffer (the main components of which were Na₂HPO₄, KH₂PO₄, NaCl, and KCl). For example, the trypsin used was TrypLE Express.

[0055] In the above embodiments, the ratio of tumor cells to gel is appropriate, which is helpful for the culture of organoids; at the same time, trypsin can effectively digest and lyse the organoids, while PBS buffer can quickly terminate the digestion and lysis process of the organoids, and the control method is relatively simple.

[0056] In one specific implementation, the fabrication method of the microfluidic chip is as follows: Microvalve control layer 1: First, using an N-type 4-inch silicon wafer, the planar structure of the microvalve control layer 1 is etched using SU-8 photoresist. Then, grooves of corresponding depth are etched using ICP dry etching to form a mold for fabricating the microvalve control layer 1. Next, liquid PDMS is poured into the mold of the microvalve control layer 1, solidified, demolded, and cut to form the microvalve control layer 1.

[0057] Processing layer 3: Using an N-type 4-inch silicon wafer, the planar structure of processing layer 3 is lithographically created using SU-8 photoresist. ICP dry etching is then used to create grooves of corresponding depths, with different heights for the grooves in different areas of processing layer 3. A second photolithography process is then used to form a mold for fabricating processing layer 3. Liquid PDMS is poured into the mold, solidifies, and then removed from the mold and cut to form processing layer 3.

[0058] Microvalve film layer 2: PDMS gel is applied to a 4-inch N-type silicon wafer on a spin coater, and a 15-micron thick PDMS film is obtained by controlling the rotation speed. Then it is placed in an oven for heating and solidification. After demolding from the silicon wafer, an elastic microvalve film layer 2 is obtained.

[0059] Chip assembly and bonding: After ultrasonic cleaning, the microvalve control layer 1, microvalve thin film layer 2, and processing layer 3 are treated with a Plasma machine, and the three are bonded together in sequence. After high-temperature baking, a complete microfluidic chip is formed.

[0060] It should be noted that the chip must be sterilized before use by immersing it in 75% alcohol and filling the chip with alcohol through the flow channels using a syringe. After standing for 6 hours, wash with PBS to remove residual alcohol. Then, expose to ultraviolet light overnight before use in subsequent experiments.

[0061] It is understood that the above embodiments are merely exemplary implementations used to illustrate the principles of the present invention, and the present invention is not limited thereto. For those skilled in the art, various modifications and improvements can be made without departing from the spirit and essence of the present invention, and these modifications and improvements are also considered to be within the scope of protection of the present invention.

Claims

1. A microfluidic chip for organoid culture and detection, characterized in that, It includes a microvalve control layer, a microvalve film layer, and a processing layer. The microvalve film layer is disposed between the microvalve control layer and the processing layer, and adjacent layers are sealed together. The microvalve control layer is provided with a first liquid inlet, a first liquid outlet, a digestive fluid inlet, a second liquid inlet, a cell suspension outlet, an amplification product outlet, a microvalve air inlet, and a microvalve control structure; wherein the first liquid inlet, the first liquid outlet, the digestive fluid inlet, the second liquid inlet, the cell suspension outlet, and the amplification product outlet all penetrate into the processing layer; the microvalve control structure includes independent gas channels, each gas channel being connected to a microvalve air inlet for controlling the gas pressure within the gas channel; the gas channels cooperate with the microvalve film layer to control the opening or closing of each microvalve control area; The processing layer has an organoid culture structure, a single-cell capture and amplification structure, and a microvalve control region on the side facing the microvalve film layer. The organoid culture structure and the single-cell capture and amplification structure each have multiple microvalve control regions. The organoid culture structure is connected to the single-cell capture and amplification structure. The organoid culture structure is connected to the first liquid inlet, the first liquid outlet, and the digestive fluid inlet. The single-cell capture and amplification structure is connected to the second liquid inlet, the cell suspension outlet, and the amplification product outlet. By controlling the opening or closing of the corresponding micro-valve control area, the gel mixture formed by mixing tumor cells and gel can enter the organoid culture structure through the first liquid inlet to culture the tumor cells, and can be discharged through the first liquid outlet; organoid digestive enzymes can enter the organoid culture structure through the digestive fluid inlet and digest the organoid mass into single cells to form a single-cell suspension; the single-cell suspension enters the single-cell capture and amplification structure from the organoid culture structure to capture single cells; the cell lysis solution entering through the second liquid inlet lyses the single cells to form cell lysis fluid, and then the amplification enzyme solution entering the reaction fluid channel through the second liquid inlet amplifies the cell lysis fluid to form amplification products, and can be discharged through the amplification product outlet; The gas channels include a first gas channel, a second gas channel, a third gas channel, a fourth gas channel, a fifth gas channel, a sixth gas channel, and a seventh gas channel; The first gas channel is equipped with a control microvalve A; the second gas channel is equipped with a control microvalve B; the third gas channel is equipped with control microvalve C, D, and E; the fourth gas channel is equipped with control microvalve F and G; the fifth gas channel is equipped with a control microvalve H; the sixth gas channel is equipped with a control microvalve I; and the seventh gas channel is equipped with a control microvalve J. Each control microvalve corresponds to a microvalve control area on the processing layer, and each control microvalve cooperates with the microvalve film layer to control the opening or closing of each microvalve control area. The organoid culture structure includes a first liquid channel, an organoid culture unit, and a digestive fluid channel, wherein one end of the first liquid channel is connected to a first liquid inlet, and the other end is connected to a first liquid outlet; the digestive fluid channel is connected to the digestive fluid inlet. The organoid culture unit includes an organoid culture chamber, a digestion mixing zone, and an organoid lysis chamber. The organoid culture chamber is connected to a first liquid channel and a digestive fluid channel, respectively. The first liquid channel is provided with a microvalve control area A for controlling the opening or closing of the first liquid channel. The digestive fluid channel is provided with a microvalve control area B for controlling the opening or closing of the digestive fluid channel. When the A microvalve control area is opened, the gel mixture formed by the mixing of tumor cells and gel enters the first liquid channel through the first liquid inlet and then enters the organoid culture chamber. Culture medium is then injected into the first liquid channel through the first liquid inlet to culture the tumor cells. When microvalve A is closed and microvalve B is opened, the organoid digestive enzymes that enter the digestive fluid channel through the digestive fluid inlet pass through the organoid culture chamber and the digestion mixing zone in sequence and enter the organoid lysis chamber. The gel mixture mixes with the organoid digestive enzymes in the digestion mixing zone and digests the organoid mass into single cells in the organoid lysis chamber to form a single-cell suspension in the organoid lysis chamber. The single-cell capture and amplification structure includes a main channel, a reaction liquid channel, and a cell processing unit. Each cell processing unit includes a cell lysis chamber, a lysis termination chamber, and an amplification chamber. One end of the main channel is connected to the organoid lysis chamber, and the other end is connected to the cell suspension outlet. The main channel includes a capture region and a bypass region, which are connected in parallel. The capture region includes an inlet end, a capture segment, a connecting segment, and an outlet end, which are sequentially connected. The bypass region, the cell lysis chamber, the termination lysis chamber, the amplification chamber, and the amplification product outlet are sequentially connected, and an F microvalve control area is provided between the bypass region and the cell lysis chamber. An H microvalve control area is provided between the cell lysis chamber and the termination lysis chamber. An I microvalve control area is provided between the termination lysis chamber and the amplification chamber. A G microvalve control area is provided between the amplification chamber and the amplification product outlet. The main channel is provided with a C-microvalve control area, a D-microvalve control area, and an E-microvalve control area. The C-microvalve control area is located at the inlet end of the main channel near the bypass area and the capture area, and is used to control the opening or closing of the main channel. The D-microvalve control area is located in the bypass area, and is situated between the outlet end of the bypass area and the connection point between the bypass area and the cell lysis chamber, and is used to control the opening or closing of the bypass area. The E-microvalve control area is located at the outlet end of the main channel near the bypass area and the capture area, and is used to control the opening or closing of the main channel. One end of the reaction liquid flow channel is connected to the second liquid inlet, and the other end is connected to the capture area. The connection point between the reaction liquid flow channel and the capture area is located in the connecting section. A sixth control valve area is provided on the reaction liquid flow channel to control the opening or closing of the reaction liquid flow channel. Opening the sixth control valve area closes the C micro valve control area, the D micro valve control area, and the E micro valve control area. When the F microvalve control area and the G microvalve control area are closed, the single-cell suspension enters the main channel from the digestive fluid channel. When it flows through the capture area, the single cell is captured and the capture area is blocked. Then the single-cell suspension flows from the bypass area to the cell suspension outlet. When the C microvalve control area and the D microvalve control area are closed, and the F microvalve control area and the G microvalve control area are opened, the cell lysis solution entering through the second liquid inlet passes through the reaction liquid channel and the capture area, carrying the captured single cells through the bypass area and into the cell lysis chamber. The F and H microvalve control areas are closed, and single cells lyse in the cell lysis chamber. The F and H microvalve control areas are then opened, allowing the lysis termination solution entering the reaction channel through the second liquid inlet to flow into the lysis termination chamber along with the liquid in the cell lysis chamber. The F and I microvalve control areas are then closed, allowing the lysis termination solution to mix thoroughly with the cell lysis liquid, thus terminating lysis. The F and I microvalve control areas are then opened, allowing the amplification enzyme solution entering the reaction channel through the second liquid inlet to flow into the amplification chamber along with the lysis termination solution. The F and J microvalve control areas are then closed, and amplification begins. After a preset time, amplification is terminated to form amplification products. Finally, the F and J microvalve control areas are opened, allowing purified water entering the reaction channel through the second liquid inlet to discharge the amplification products from the amplification product outlet. The diameter of the main channel and the channel through which single cells can pass is 30 micrometers, and the diameter of the trapping section is 5 micrometers; The gel mixture is injected through the first liquid inlet and can be injected into the organoid culture chamber under the action of gravity. The microfluidic chip is placed in a 37°C incubator for half an hour to fix the gel. Then, sterile gas is injected through the first liquid inlet to remove the residual gel mixture in the first liquid channel.

2. The microfluidic chip for organoid culture and detection according to claim 1, wherein, Multiple cell processing units are connected in parallel, each cell processing unit corresponds to a capture area and a bypass area, and the E-microvalve control area is provided between adjacent capture areas.

3. The microfluidic chip for organoid culture and detection according to claim 1, wherein, The number of organoid culture units and digestive fluid channels are both multiple. Multiple organoid culture units are connected in series through the first liquid channel. The A microvalve control area is set between adjacent organoid culture units. Each organoid culture unit corresponds to one digestive fluid channel.

4. The microfluidic chip for organoid culture and detection according to claim 1, wherein, The microvalve control layer, microvalve film layer, and processing layer are all made of PDMS material. The microvalve control layer and the microvalve film layer are bonded together using oxygen plasma assisted bonding, and the microvalve film layer and the processing layer are bonded together using oxygen plasma assisted bonding.

5. The microfluidic chip for organoid culture and detection according to claim 4, wherein, The microvalve film layer is spin-coated from PDMS material and has a thickness of 15 micrometers.

6. The microfluidic chip for organoid culture and detection according to claim 2, wherein, The main channel is bent multiple times to form multiple interconnected sub-channels, each of which is connected to multiple cell processing units.

7. The microfluidic chip for organoid culture and detection according to claim 6, wherein, The reaction liquid channel includes multiple sub-reaction liquid channels, one end of which is connected to the second liquid inlet, and the other end of which is connected to a capture area.

Images