Micro-fluidic chip for fixedly clamping organoid globules and preparation method of micro-fluidic chip

Abstract

The invention provides a micro-fluidic chip for fixedly clamping a globule like an organ. The micro-fluidic chip comprises a micro-channel structure, the micro-channel structure comprises an air cavity structure located on the upper layer and a clamping channel located on the lower layer. The air cavity structure forms a closed cavity and is used for applying pressure to the clamping channel; a clamping structure is arranged in the clamping channel and is of a claw-shaped structure composed of a plurality of cones. The micro-fluidic chip disclosed by the invention can stably fix the organoid globule, prevent the organoid globule from floating away from a detection surface and ensure the stability of a cell position in a signal acquisition process, so that continuous and stable acquisition of signals can be ensured in time and space, and the continuity and accuracy of signal acquisition are improved; fresh culture solution is allowed to flow, nutrients are provided for the organ-like pellets, and long-term activity and functions of the organ-like pellets are maintained; in addition, pollution and interference caused by chemical molecules are not introduced, and the organ-like small balls can be released and recycled at any time as required.

Description

Technical Field

[0001] The present invention relates to the technical field of microfluidic chips, and in particular to a microfluidic chip for fixing and clamping organoid pellets and a preparation method thereof. Background Art

[0002] Organoids have been a hot topic in recent years and have developed rapidly. They are of great significance in both basic and clinical research. Organoids have advantages over traditional 2D culture and can show cell composition and behavior close to physiology. The emergence of organoid research no longer restricts medical research. By studying the response of organoids to external stimuli, we can have a deeper understanding of the functions and physiological mechanisms of real organs, simulate the development of diseases, and provide new ideas and methods for the treatment of diseases. This is of great significance for understanding cellular processes, disease mechanisms, and drug development.

[0003] Microelectrode array (MEA) is a commonly used electrical detection technology for recording electrophysiological activity of organoid spheres. It can be used to monitor the electrical activity of neurons in organoids. MEA is a method that uses arrayed electrodes to record electrical signals from discharge cells. These electrodes can capture the weak electrical signals generated by cells during activity, thereby enabling real-time monitoring of cell activity. By analyzing these electrical signals, researchers can understand the electrical response of organoids to external stimuli.

[0004] In the step of obtaining electrical signals of organoid cells in traditional MEA technology, before placing organoid cells on MEA, MEA needs to be pre-treated and coated with adhesion laminin for 30 minutes to ensure that its surface is suitable for cell growth and adhesion. Organoid cells are placed on top of the electrode array in an appropriate manner, and the cell's own adhesion and adhesion molecules are used to promote the fit between the cell and MEA. The MEA pretreatment operation is mainly to make the cell and the electrode form a closer fit and obtain cell biological signals. The size and shape of the recorded signal depends on several factors: the nature of the contact between the cell and the MEA electrode (such as contact area and tightness); the voltage amplitude detected by the electrode is inversely proportional to the distance of cell depolarization, so it is necessary to culture cells on the electrode to place the cells as close to the electrode as possible.

[0005] The following problems may occur in the process of using the MEA method to process cells to obtain electrical signals: 1. The adhesion effect between the organoid pellet and the electrode array is not obvious, and the organoid cells cannot be firmly attached to the electrode surface. This will result in leakage current, which will introduce noise and reduce the accuracy and reliability of the measurement, which will seriously interfere with the measurement of cell bioelectrical characteristics; 2. The cell position is not firmly fixed and deviates from the detection position. When the cell culture fluid is perfused, the organoid pellet may drift away from the detection surface, and the cell position cannot be fixed, resulting in detection interruption and long-term effective detection.

[0006] In view of this, the present invention is proposed. Summary of the invention

[0007] The object of the present invention is to provide a microfluidic chip for fixing and clamping organoid pellets and a preparation method thereof, which can stably fix the organoid pellets on the detection surface of the biosensor and realize the perfusion culture of cells to improve the stability of signal acquisition and the efficiency of cell culture.

[0008] According to a first aspect of the present invention, there is provided a microfluidic chip for fixing and clamping organoid pellets, comprising: a microchannel structure; the microchannel structure comprises: an air cavity structure located at an upper layer and a clamping channel located at a lower layer; the air cavity structure forms a closed cavity for applying pressure to the clamping channel; a clamping structure is arranged in the clamping channel, and the clamping structure is a claw-shaped structure composed of a plurality of cones.

[0009] Preferably, two ends of the microchannel structure are provided with a sample inlet and a sample outlet, and the sample inlet and the sample outlet are respectively communicated with the clamping channel.

[0010] Preferably, the clamping structure is located in the middle of the clamping channel, and the air cavity structure is located above the clamping structure.

[0011] Preferably, a plurality of the cones form a space adapted to the size of the organoid pellet, ensuring that the organoid pellet can be fixed in the clamping channel.

[0012] Preferably, the number of the cones is four.

[0013] Preferably, it also includes a glass electrode bonded to the microchannel structure.

[0014] Preferably, the clamping channel is a straight channel with one inlet and one outlet. When in use, the liquid level difference between the inlet and outlet holes drives the organoid pellet to deform into the clamping area and be stably clamped for culture and detection.

[0015] Preferably, the material of the microchannel structure is formed by mixing PDMS monomer and curing cross-linking agent; more preferably, PDMS monomer and curing cross-linking agent are mixed in a mass ratio of (8-12): 1. The selection of PDMS as the main material of the microfluidic chip is mainly based on the following advantages: first, it has high transparency and is easy to observe; second, PDMS treated with oxygen plasma can form irreversible and strong chemical bonds on the glass surface, and the bonding process is fast and can be completed in only 3 minutes; more importantly, PDMS has good air permeability, which can effectively ensure the exchange of gas and nutrients during cell culture and improve cell culture efficiency.

[0016] A second aspect of the present invention provides a method for preparing a microfluidic chip for fixing and clamping organoid pellets, comprising the following steps:

[0017] S1. preparing a 3D printed microchannel structure mold;

[0018] S2, after mixing the PDMS monomer and the curing cross-linking agent, place them in a vacuum device to exhaust the gas, pour them on the 3D printed microchannel structure mold, and place them in an oven to cure the PDMS;

[0019] S3. Peeling the solidified PDMS off the mold to obtain a microchannel structure, and punching inlet holes and outlet holes at both ends of the microchannel structure.

[0020] Preferably, the method further comprises the following steps:

[0021] S4. Place the microchannel structure and the glass electrode in a plasma processor. The microchannel structure treated with oxygen plasma will be bonded to the surface of the glass electrode. The Si-OH groups on the surfaces of both will undergo a dehydration reaction to form Si-O-Si bonds. The formation of chemical bonds is the key to achieving a strong bond between PDMS and the glass electrode, making the chip bond tighter, creating a microchannel with cell-enclosed and sensor-close contact, and completing the production of the microfluidic chip.

[0022] A third aspect of the present invention provides a microfluidic system, comprising: a perfusion system and the above-mentioned microfluidic chip for fixing and clamping organoid pellets, wherein the perfusion system is connected to the microfluidic chip.

[0023] Preferably, the perfusion system comprises: a syringe pump and a control system, the syringe pump is connected to the microfluidic chip through a pipeline, and the control system is used to control the operation of the syringe pump.

[0024] The present invention has at least the following beneficial effects:

[0025] (1) The microfluidic chip of the present invention is used for a biosensor. The clamping structure and the air cavity structure in the clamping channel can stably fix the organoid pellet on the detection surface of the base in the microfluidic chip, ensure the stability of the cell position during signal acquisition, prevent the organoid pellet from drifting away from the detection surface, and prevent the cell drifting and rotation from causing changes in the corresponding detected position of the organoid pellet in contact with the sensor, making it possible to stably observe the multi-point spatial characteristics of the organoid pellet surface over time or change over time, thereby achieving continuous and stable signal acquisition in both the time dimension and the space dimension, and improving the continuity and accuracy of signal acquisition.

[0026] (2) The microfluidic chip of the present invention takes into account the perfusion culture requirements of cells. Fresh culture fluid is allowed to flow in the clamping channel to maintain cell activity. The clamping structure clamps the organoid pellet in the center of the clamping channel, occupying the middle part in the cross section. The perfused culture fluid can flow from both sides and above the organoid pellet, providing nutrients for the organoid pellet and discharging waste fluid, thereby maintaining the long-term activity and function of the organoid pellet.

[0027] (3) The microfluidic chip of the present invention adopts physical clamping, which will not introduce contamination and interference of chemical molecules, and can release and recover organoid cell pellets whenever needed.

[0028] (4) The microfluidic chip of the present invention is made of conventional materials such as PDMS and can be reused. In addition, the microfluidic system is simple in structure and does not require external peripherals, thus having the advantage of low cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0030] Figure 1 A schematic diagram of the structure of a microfluidic chip for fixing and clamping organoid pellets provided by the present invention;

[0031] Figure 2 A top view of a microfluidic chip for fixing and clamping organoid pellets provided by the present invention;

[0032] Figure 3 A cross-sectional view of a microfluidic chip for fixing and clamping organoid pellets provided by the present invention;

[0033] Figure 4 This is a schematic diagram of the structure of the microfluidic system provided by the present invention.

[0034] Explanation of the accompanying drawings: 1. microchannel structure; 2. glass electrode; 3. air cavity structure; 4. clamping channel; 5. sample inlet; 6. sample outlet; 7. clamping structure; 8. myocardial organoid pellet; 9. injection pump. DETAILED DESCRIPTION

[0035] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0036] In the description of the present invention, it should be understood that the terms "center", "longitudinal", "lateral", "length", "width", "thickness", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside", "clockwise", "counterclockwise" and the like indicate orientations or positional relationships based on the orientations or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the referred device or element must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be understood as limiting the present invention.

[0037] In addition, the terms "first" and "second" are only used for descriptive purposes and cannot be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. In the description of the present invention, "multiple" means two or more, unless otherwise clearly and specifically defined. In addition, the terms "installed", "connected" and "connected" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection, or it can be indirectly connected through an intermediate medium, or it can be the internal connection of two components. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0038] Example 1

[0039] like Figures 1 to 3 As shown, this embodiment provides a microfluidic chip for fixing and clamping organoid pellets, including: a microchannel structure 1 and a glass electrode 2 bonded to the microchannel structure; the microchannel structure 1 is divided into two layers, including: an air cavity structure 3 located in the upper layer and a clamping channel 4 located in the lower layer; the air cavity structure 3 forms a closed cavity for applying pressure to the clamping channel 4; a clamping structure 7 is arranged in the clamping channel 4, and the clamping structure 7 is a claw-shaped structure composed of a plurality of cones.

[0040] In this embodiment, the two ends of the microchannel structure 1 are provided with an inlet hole 5 and an outlet hole 6, which are respectively connected to the clamping channel 4. When the clamping channel 4 is used with one inlet and one outlet, the liquid level difference between the inlet hole 5 and the outlet hole 6 drives the organoid pellet to deform into the clamping area and be stably clamped for culture and inspection.

[0041] In this embodiment, the clamping structure 7 is located in the middle of the clamping channel 4, and the air cavity structure 3 is located above the clamping structure 7. By applying additional pressure to the air cavity structure 3, the position of the clamping channel 4 can be controlled to press downward, exerting a more stable and greater clamping force on the organoid pellet. This "downward pressure" effect not only enhances the stability of the clamping, but also significantly improves the ability to capture the organoid pellet, ensuring a higher clamping efficiency of the clamping channel 4 and more reliable experimental results.

[0042] In this embodiment, a plurality of cones form a space adapted to the size of the organoid pellet, ensuring that the organoid pellet can be fixed in the clamping channel 4 .

[0043] In this embodiment, there are four cones, and the spacing between the four cones is determined by the diameter of the organoid pellet. The size is slightly smaller than the diameter of the organoid pellet. The curvature of the sphere is subtracted from the middle of the cone shape to ensure that the organoid pellet can be stably embedded in it and is not easy to fall off.

[0044] In this embodiment, the clamping channel 4 is a straight channel. During operation, the organoid pellets only need to be controlled by the flow rate to enter the clamping area, and the organoid pellets can be released from the clamping position by changing the flow rate. The size of the clamping channel 4 is adjusted according to the size of the organoid pellets to ensure that the pellets can be stably clamped in the channel.

[0045] In this embodiment, the material of the microchannel structure 1 is formed by mixing PDMS monomer and a curing cross-linking agent.

[0046] This embodiment also provides a method for preparing a microfluidic chip for fixing and clamping organoid pellets, comprising the following steps:

[0047] S1. preparing a 3D printed microchannel structure mold;

[0048] Specifically, a channel with a claw-shaped clamping structure can be designed using 3D design technology, and it can be precisely manufactured using 3D printing technology. The clamping structure 7 is designed using 3D modeling software, and the height and spacing of the clamping structure 7 are related to the size and diameter of the organoid sphere;

[0049] S2, mixing PDMS monomer and curing cross-linking agent in a mass ratio of 10:1, stirring evenly and placing in a vacuum device to exhaust gas, pouring on a 3D printed microchannel structure mold, and placing in an oven to cure PDMS;

[0050] S3, peeling the solidified PDMS from the mold to obtain a microchannel structure 1, and punching a sample inlet hole 5 and a sample outlet hole 6 at both ends of the microchannel structure 1;

[0051] S4, placing the microchannel structure 1 and the glass electrode 2 in a plasma processor, and bonding the surfaces of the microchannel structure 1 and the glass electrode 2 treated with oxygen plasma to complete the production of the microfluidic chip.

[0052] When in use, the microfluidic chip is connected to the perfusion system, and the organoid pellets are transported to the clamping channel 4 through the injection pump 9 and pipeline of the perfusion system; the injection pump 9 transports the culture fluid at a set flow rate and flow rate for cell perfusion culture; to the cell clamping area, according to the pressure difference between the sample inlet 5 and the sample outlet 6, the organoid pellets are clamped in the clamping structure 7, which stably increases the contact area between the cells and the glass electrode 2. During this process, the sensor electrode array located below the clamping position can accurately collect the weak signals generated by the cells.

[0053] Example 2

[0054] like Figure 4 As shown, this embodiment provides a microfluidic system, including: a perfusion system and a microfluidic chip for fixing and clamping organoid beads of Example 1, and the perfusion system is connected to the microfluidic chip.

[0055] In this embodiment, the perfusion system includes: an injection pump 9 and a control system. The injection pump 9 is connected to the microfluidic chip through a pipeline, and the control system is used to control the operation of the injection pump 9.

[0056] Specifically, the perfusion system can be composed of an injection pump 9, a transmission device, and a control system pipeline integrated perfusion system. The injection pump 9 is added with the organoid pellet culture fluid and connected to the sampling hole 5 of the microfluidic chip to ensure the continuous flow of fresh culture fluid; the fresh culture fluid is added to the syringe, and the injection pump 9 delivers the culture fluid to the cell culture area at a set flow rate and flow rate through the pump and pipeline of the perfusion system. The perfusion rate and perfusion time can be adjusted as needed to meet the growth requirements of the cells; a liquid storage tank is inserted at the sampling hole 6 to achieve continuous perfusion culture.

[0057] Test example

[0058] This detection example uses the microfluidic chip of Example 1 and the microfluidic system of Example 2 to achieve stable detection of electrophysiological signals of myocardial organoid spheres 8. First, the chips, instruments and reagents used are sterilized, and then the flow channel circuit of the chip is built using the sterilized equipment.

[0059] In the perfusion system, a syringe pump 9 is used to provide power to pump culture fluid into the cell culture chamber and discharge waste fluid through the microchannel structure 1 made in the chip. The cardiomyocytes used in the experiment are injected into the chip chamber from the cell injection hole 5 of the chip using a syringe, and the cardiomyocytes in the chip are continuously perfused and cultured using a syringe pump 9. The culture fluid is continuously and stably input into the chip from the flow channel injection hole 5 at a fixed flow rate to ensure that the organoid pellets can be stably clamped.

[0060] In the acquisition system, a MEA with 100 electrodes is used to form a sensor for collecting cell signals. Myocardial cells are cells with autonomy and excitability, which can spontaneously generate electrical signals. The microelectrode array collects the potential changes generated by myocardial cells on the electrodes, collects the weak signals generated by myocardial cells, inputs them into the acquisition card, and converts them into digital signals through the biosensor platform and transmits them wirelessly to the computer, where they are filtered and displayed. The test results show that the stable biological signals of myocardial cell beating can be clearly seen.

[0061] In summary, the microfluidic chip of the present invention is used for biosensors, and the clamping structure 4 and the air cavity structure 3 in the clamping channel 4 can stably fix the organoid sphere on the detection surface of the base in the microfluidic chip, ensure the stability of the cell position during signal acquisition, prevent the organoid sphere from drifting away from the detection surface, and prevent the cell from drifting and rotating to cause the corresponding detected position of the organoid sphere in contact with the sensor to change, so that it is possible to stably observe the multi-point spatial characteristics of the surface of the organoid sphere for a long time to maintain or change over time, thereby achieving continuous and stable signal acquisition in both the time dimension and the space dimension, and improving the continuity and accuracy of signal acquisition. The microfluidic chip of the present invention takes into account the perfusion culture requirements of cells, and allows fresh culture fluid to flow in the clamping channel 4 to maintain cell activity; the clamping structure clamps the organoid sphere in the center of the clamping channel, and occupies the middle part in the cross section. The perfused culture fluid can flow from both sides and above the organoid sphere, providing nutrients for the organoid sphere, discharging waste fluid, and maintaining the long-term activity and function of the organoid sphere. The microfluidic chip of the present invention adopts physical clamping, which will not introduce pollution and interference of chemical molecules, and can release and recover organoid cell pellets at any time. The microfluidic chip of the present invention is made of conventional PDMS and other materials, which can be reused, and the microfluidic system is simple in structure, does not require external peripherals, and has the advantage of low cost.

[0062] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. 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 replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A microfluidic chip for fixing and clamping organoid pellets, characterized in that: include: Microchannel structure; the microchannel structure includes: an air cavity structure located at the upper layer and a clamping channel located at the lower layer; the air cavity structure forms a closed cavity for applying pressure to the clamping channel; a clamping structure is arranged in the clamping channel, and the clamping structure is a claw-shaped structure composed of a plurality of cones.

2. The microfluidic chip for fixing and clamping organoid pellets according to claim 1, characterized in that: The two ends of the microchannel structure are provided with a sample inlet and a sample outlet, and the sample inlet and the sample outlet are communicated with the clamping channel respectively.

3. The microfluidic chip for fixing and clamping organoid pellets according to claim 1, characterized in that: The clamping structure is located in the middle of the clamping channel, the air cavity structure is located above the clamping structure, and the clamping channel is a straight channel.

4. The microfluidic chip for fixing and clamping organoid pellets according to claim 1, characterized in that: A plurality of the cones form a space adapted to the size of the organoid pellet, ensuring that the organoid pellet can be fixed in the clamping channel.

5. The microfluidic chip for fixing and clamping organoid pellets according to claim 1, characterized in that: The number of the cones is four.

6. The microfluidic chip for fixing and clamping organoid pellets according to claim 1, characterized in that: Also included is a glass electrode bonded to the microchannel structure.

7. The method for preparing a microfluidic chip for fixing and clamping organoid beads according to any one of claims 1 to 6, characterized in that: The steps include: S1. Prepare a 3D printed microchannel structure mold; S2, after mixing the PDMS monomer and the curing cross-linking agent, place them in a vacuum device to exhaust the gas, pour them on the 3D printed microchannel structure mold, and place them in an oven to cure the PDMS; S3. Peeling the solidified PDMS off the mold to obtain a microchannel structure, and punching inlet holes and outlet holes at both ends of the microchannel structure.

8. The method for preparing a microfluidic chip for fixing and clamping organoid beads according to claim 7, characterized in that: The following steps are also included: S4. Place the microchannel structure and the glass electrode in a plasma processor, and adhere the microchannel structure treated with oxygen plasma to the surface of the glass electrode.

9. A microfluidic system, characterized in that: include: A perfusion system and a microfluidic chip for fixing and clamping organoid pellets as described in any one of claims 1 to 6, wherein the perfusion system is connected to the microfluidic chip.

10. The microfluidic system according to claim 9, characterized in that: The perfusion system comprises: an injection pump and a control system. The injection pump is connected to the microfluidic chip through a pipeline, and the control system is used to control the operation of the injection pump.

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